Compositions and methods for delivering lypophilic agents to dental pulp and for enhancing dentin production

ABSTRACT

Methods and compositions are provided for enhancing dentin production, and for delivering a lipophilic agent to pulp tissue of a tooth of an individual. In some cases, a subject method includes a step of administering to the pulp of a tooth of an individual, a Wnt stimulating composition that includes a Wnt stimulator agent, at a dose sufficient to enhance the production of dentin by the pulp. In some cases, a subject method includes a step of contacting exposed dentin of a tooth with a composition that includes a lipophilic agent inserted in the non-aqueous phase of a lipid structure (e.g., whereby the lipophilic agent penetrates the dentin to the underlying pulp tissue). Kits are also provided for practicing the methods of the disclosure.

CROSS REFERENCE

This application claims benefit and is a Continuation of 371 applicationSer. No. 15/539,489, filed Jun. 23, 2017, which claims benefit of PCTApplication No. PCT/US2015/067683, filed Dec. 28, 2015, which claimsbenefit of U.S. Provisional Patent Application No. 62/097,502, filedDec. 29, 2014, which applications are incorporated herein by referencein their entirety.

INTRODUCTION

Many toothaches are the result of chronic bacterial infections thatcause inflammation of the connective, vascular, lymphatic and nervoustissues occupying a chamber in the center of the tooth. When thesetissues, collectively referred to as the pulp, become chronicallyinflamed they must be removed in a procedure known as a root canaltreatment. Even when bacterial infections do not penetrate into the pulpchamber, a root canal may be required because bacterial by-products candiffuse through the remaining tooth structure and cause chronic pulpinflammation. In an effort to treat these conditions, a century's oldprocedure called pulp capping is often employed, which consists ofplacing a material such as calcium hydroxide on the remaining toothstructure that creates a high pH, antimicrobial environment.

Tooth sensitivity affects millions of people and can be traced to aninadequate amount of dentin insulating the pulp cavity. Normally, dentinis insulated by enamel on the crown of the tooth, and by gum tissue onthe roots of the teeth. Tooth sensitivity can occur when theseinsulators deteriorate. For example, tooth sensitivity can arise becauseof a deep cavity, a deep dental restoration (e.g., amalgam, composite, acrown, etc.), periodontal disease, or because of aging.

One goal of regenerative dental medicine is to stimulate the generationof dentin (e.g., from odontoblasts) with the same structural andbiological properties of native dentin. In doing so, the vitality andfunction of the existing teeth can be preserved. Odontoblasts secrete anextracellular matrix that undergoes mineralization and are trappedwithin the pulp chamber. Unless the pulp cavity has been exposed,delivery of medicants (e.g., therapeutic agents) to pulpal tissues isdifficult. The pulp is surrounded by dentin, a mineralized matrix thatprotects the pulp cavity from thermal, chemical and other noxiousstimuli. The only means to access the pulp is either through mechanicalexposure (drilling) or delivery of the medicant via the dentinaltubules. Dentinal tubules are small (2.5 μm diameter), fluid filledcannuli that house the odontoblastic process.

The present disclosure provides compositions and methods for deliveringlipophilic agents to pulp tissue, and/or for enhancing dentin productionby dental pulp tissue (e.g., in the context of pulp exposure, toothsensitivity, and the like).

PUBLICATIONS

Han et al., PLoS One. 2014 Feb. 10; 9(2):e88890; Yang and Liu, StemCells In Oral Medicine. 2012; 1(1): 3-8; Arioka et al., BiochemPharmacol. 2014 Aug. 15; 90(4):397-405; Biomaterials. 2015 January;39:145-54, Epub 2014 Nov. 22; Thesleff and Tummers, StemBook [Internet].Cambridge (Mass.): Harvard Stem Cell Institute; 2008-2009 Jan. 31;Minear et al., Sci Transl Med. 2010 Apr. 28; 2(29):29ra30; Westendorf etal., Gene. 2004 Oct. 27; 341:19-39; Moon et. al., Nat Rev Genet. 2004September; 5(9):691-701; Dhamdhere et al., PLoS One. 2014 Jan. 6;9(1):e83650; Zhao et al., Methods Enzymol. 2009; 465:331-47; U.S. patentpublication numbers: 20140371151, 20120115788, 20120329790, 20120231091,and 20080226707; PCT publication number WO2012122081; and U.S. Pat. No.8,809,272.

SUMMARY

Methods and compositions are provided for enhancing dentin production,and for delivering a lipophilic agent to pulp tissue of a tooth of anindividual. In some embodiments, a subject method includes a step ofadministering to the pulp of a tooth of an individual, a Wnt stimulatingcomposition that includes a Wnt stimulator agent, at a dose sufficientto enhance the production of dentin by the pulp. In some cases, the pulpis exposed pulp and the administering step includes contacting theexposed pulp with the Wnt stimulating composition. In some cases, theadministering step includes a step of contacting dentin with the Wntstimulating composition, whereby the Wnt stimulating compositionpenetrates the dentin to the underlying pulp tissue. In some cases, theWnt stimulating composition includes a lipophilic Wnt stimulator agentinserted in the non-aqueous phase of a lipid structure. In some suchcases, the lipophilic Wnt stimulator agent is a Wnt protein (e.g., a Wntprotein having a lipid moiety). In some cases, the Wnt protein is Wnt3A(e.g., human Wnt3A). Thus, in some cases, the Wnt stimulatingcomposition includes a liposomal Wnt (L-Wnt), e.g., liposomal Wnt3A(L-Wnt3A).

In some embodiments, a subject method includes a step of contactingexposed dentin of a tooth with a composition that includes a lipophilicagent inserted in the non-aqueous phase of a lipid structure (e.g.,whereby the lipophilic agent penetrates the dentin to the underlyingpulp tissue). In some cases, the individual has tooth sensitivity or isat risk of developing tooth sensitivity (e.g., following a dentalprocedure). In some cases, the pulp of the tooth is exposed and in somecases, the pulp of the tooth is not exposed. In some cases, a subjectmethod includes, prior to the contacting step, a step of exposing dentinof the tooth. In some cases, the lipophilic agent is a growth factorhaving a lipid moiety. In some cases, the lipophilic agent is a Wntstimulator agent having a lipid moiety. In some such cases, the Wntstimulator agent is a Wnt protein (e.g., a Wnt protein having a lipidmoiety). In some cases, the Wnt protein is Wnt3A (e.g., human Wnt3A).Thus, in some cases, the lipophilic agent inserted in the non-aqueousphase of a lipid structure is a liposomal Wnt (L-Wnt), e.g., liposomalWnt3A (L-Wnt3A).

In some cases, a Wnt3A protein is delivered to the pulp cavity. Thelipophilic WNT3A protein can be tethered to a lipid vesicle, which canstabilize the in vivo biological activity of the protein. The liposomalWnt3A (L-Wnt3A) formulation can be applied to exposed dentin, wherebythe liposomal particles penetrate the dentin through the dentinaltubules, which extend from the outside of the tooth to the pulp cavity.There, L-Wnt3A can enhance both the survival and proliferation of pulpcells, and can stimulate the formation of dentin (e.g., tertiarydentin), which insulates the tooth and protects the pulp from thermaland chemical insult. By stimulating new dentin formation, the risk ofbacterial infection of the pulp and overall tooth sensitivity arereduced and the need for root canal therapies, extensive prostheticreplacements, and tooth extraction are reduced. The subject methods canaugment the body's natural response to tooth sensitivity: topicalapplication of a liposomal protein therapeutic can stimulate dental pulpcells to produce more dentin and in doing so, provide additionalinsulation to the teeth. The subject methods have broad applications ingeneral restorative dentistry, prosthodontics, and periodontics. Kitsare provided for practicing the methods of the disclosure.

BRIEF DESCRIPTION OF THE DRAWINGS

The invention is best understood from the following detailed descriptionwhen read in conjunction with the accompanying drawings. The patent orapplication file contains at least one drawing executed in color. Copiesof this patent or patent application publication with color drawing(s)will be provided by the Office upon request and payment of the necessaryfee. It is emphasized that, according to common practice, the variousfeatures of the drawings are not to-scale. On the contrary, thedimensions of the various features are arbitrarily expanded or reducedfor clarity. Included in the drawings are the following figures.

FIG. 1A-1F. Odontoblasts are Wnt responsive FIG. 1A In skeletally maturemice, pentachrome staining identifies dentin (yellow to yellow-green),pulp (purple) and alveolar bone (yellow). FIG. 1B Higher magnificationof the pulpal-dentin complex illustrates the organization of pulp cellsand odontoblasts (pink) juxtaposed to the pre-dentin and dentin (blueand blue-yellow). FIG. 1C In the pulp cavity only polarized, secretoryodontoblasts are positive for Nestin immunostaining. FIG. 1D Thesepolarized, secretory odontoblasts express DSP. FIG. 1E X-gal stainingand FIG. 1F GFP fluorescence, respectively in adult Axin2^(LacZ/+) andAxin2^(CreERT2/+); R26R^(mTmG/+) mice, demonstrates that polarized,secretory odontoblasts and pulp cells are Wnt responsive. Abbreviations:ab, alveolar bone; d, dentin; od, odontoblast; p, pulp; pd, pre-dentin.Scale bars: 400 μm FIG. 1A, 25 μm FIG. 1B-1E, 10 μm FIG. 1F.

FIG. 2A-2P. Axin2 deletion does not disrupt odontogenesis orpulpal-dentin homeostasis. (FIG. 2A-2F) Micro-computed tomography (μCT)reconstructions of the molar region in skeletally mature, male (FIG. 2A,2C, 2E) Axin2^(LacZ/+) and (FIG. 2B, 2D, 2F) Axin2^(LacZ/LacZ) mice.Quantified μCT demonstrate no differences in (FIG. 2G) dentin volume, or(FIG. 2H) dentin and enamel mineral density in molars from age-matched,sex-matched, adult Axin2^(LacZ/+) and Axin2^(LacZ/LacZ) mice.Pentachrome staining indicates the cellularity and organization of thepulp cavities from (FIG. 2I) Axin2^(LacZ/+) and (FIG. 2J)Axin2^(LacZ/LacZ) mice. (FIG. 2K) X-gal staining in odontoblasts andsub-odontoblasts in Axin2^(LacZ/+) and (L) Axin2^(LacZ/LacZ) mice; thestronger staining in (FIG. 2L) is due to Axin2^(LacZ/LacZ) mice carryingtwo copies of the LacZgene. (FIG. 2M) Quantitative RT-PCR analyses ofpulp tissues from Axin2^(LacZ/+) and Axin2^(LacZ/LacZ) mice, evaluatedfor the relative expression levels of Left, Axin2 exon1, and PCNA. (FIG.2N) Nestin immunostaining in Axin2^(LacZ/+) and (FIG. 2P)Axin2^(LacZ/LacZ) mice. (FIG. 2P) Quantitative qRT-PCR analyses of pulptissues from Axin2^(LacZ/+) and Axin2^(LacZ/LacZ) mice, evaluated forexpression of Nestin, DSPP, OC, and Col1. Abbreviations: ab, alveolarbone; d, dentin; p, pulp. Scale bars: 500 μm (FIG. 2A-2F), and 100 μm(FIG. 2I-2L and FIG. 2N-2O).

FIG. 3A-3Q. Injury response to an acute pulp exposure inAxin2^(LacZ/LacZ) mice. (FIG. 3A) In Axin2^(LacZ/+) mice on day 14,pentachrome staining identifies a pink-colored, acellular granulationtissue that occupies the pulp injury site. (FIG. 3B) InAxin2^(LacZ/LacZ) mice, the pulp injury site is occupied by agreen-yellow mineralized matrix and a dense infiltrate of cells. (FIG.3C) Quantification of the histomorphometric analyses, demonstrating pulpinjury sites in Axin2^(LacZ/LacZ) mice. Under polarized light,Picrosirius red staining of (FIG. 3D) Axin2^(LacZ/+) injury sites and(FIG. 3E) Axin2^(LacZ/LacZ) injury sites. In tissues from Axin2^(LacZ/+)and Axin2^(LacZ/LacZ) mice respectively, immunostaining for (FIG. 3F-3G)DSP, and (FIG. 3H-3I) Nestin. Pentachrome staining of pulp injuries onpost-injury day 4 in (FIG. 3J) Axin2^(LacZ/+) and in (FIG. 3K)Axin2^(LacZ/LacZ) mice, quantified in (FIG. 3L) where Axin2 exon1expression was measured. TUNEL staining indicates programmed cell deathin (FIG. 3M) Axin2^(LacZ/+) and (FIG. 3Q) Axin2^(LacZ/LacZ) mice (FIG.3O) Quantitative RT-PCR for CASP8 expression. On post-injury day 7, Ki67immunostaining in (FIG. 3P) Axin2^(LacZ/+) and (FIG. 3Q)Axin2^(LacZ/LacZ) mice. Abbreviations: ab, alveolar bone; d, dentin; f,furcation; gr, granulation tissue; p, pulp. Scale bars: 50 μm (FIG.3K-3L), 100 μm (FIG. 3N-3O), 50 μm (FIG. 3H-3I), 100 μm (FIG. 3A-3B),100 μm (FIG. 3D-3I), 25 μm (FIG. 3N), and 50 μm (FIG. 3O). Singleasterisk denotes P<0.05. Double asterisk denotes P<0.01. Error Barsrepresent SEM.

FIG. 4A-4F. WNT3A stimulates proliferation and survival of human dentalpulp stem cells and mouse bone marrow-derived stem cells. FIG. 4AQuantitative RT-PCR analyses following 6, 12, 24 hours of L-PBS orL-WNT3A treatment of human dental pulp stem cells. FIG. 4B Twelve hourspost treatment, the proliferative capacity of hDPSCs was assayed usingthe BrdU incorporation. FIG. 4C Quantitative RT-PCR for CASP3expression. FIG. 4D Quantitative RT-PCR analyses following 6, 12, 24hours of L-PBS or L-WNT3A treatment of whole bone marrow cells. FIG. 4ETwelve hours after whole bone marrow cells were exposed to L-WNT3A andL-PBS Ki67 expression and FIG. 4F TUNEL activity were evaluated. Scalebars: 100 μm (FIG. 4B, 4E, 4F). Single asterisk denotes P<0.05. ErrorBars represent SEM.

FIG. 5A-5O. L-WNT3A treatment induces dentin regeneration. Pentachromestaining of pulp injuries on post-injury day 4 in FIG. 5A L-PBS treatedrats and in FIG. 5B L-WNT3A treated rats. Post-surgical day 4, TUNELstaining in the pulp cavities of FIG. 5C, 5C(i) L-PBS and FIG. 5D, 5D(i)L-WNT3A treated rats. On post-surgical day 4, PCNA expression in thepulp cavities of FIG. 5E L-PBS and FIG. 5F L-WNT3A treated rats.Pentachrome staining of FIG. 5G L-PBS and FIG. 5H L-WNT3A treated rats14 days after injury. FIG. 5I Quantification of reparative dentinmatrix. Under polarized light, Picrosirius red staining of pulp injurieson post-injury day 14 in FIG. 5J L-PBS and FIG. 5K L-WNT3A treatedinjury sites. On post-surgical day 14, Nestin expression in FIG. 5LL-PBS and FIG. 5M L-WNT3A treated rats, and DSP expression in FIG. 5NL-PBS and FIG. 5O L-WNT3A treated rats. Abbreviations: dentin, d; in,injury site. Scale bars: 200 μm FIG. 5A-5D, 100 μm FIG. 5C(i), 5D(i), 50μm FIG. 5E-5F, 25 μm FIG. 5G-5O, 50 μm. Single asterisk denotes P<0.01.Error Bars represent SEM.

FIG. 6A-6F. In an embryonic 18.5 mouse molar FIG. 6A pentachromestaining identifies the enamel organ (dotted lines) and dentalmesenchyme. FIG. 6B X-gal staining of a molar tooth bud from anAxin2^(LacZ/+) embryo. On an adjacent tissue section, FIG. 6C Lef1immunostaining identifies Wnt responsive cells in the outer enamel organand in the condensing dental mesenchyme. At post-natal day 7, FIG. 6Dperiodic acid schiff staining identifies polarized odontoblasts andtheir newly secreted dentin matrix (pink), which approximates newlysecreted enamel matrix (red) produced by ameloblasts. FIG. 6E Thesepolarized, secretory odontoblasts express DSP. FIG. 6F In Axin2^(LacZ/+)mice, X-gal staining demonstrates that polarized, secretory odontoblastsare Wnt responsive. Abbreviations: am, ameloblasts; ab, alveolar bone;d, dentin; df, dental follicle; dp, dental papilla; e, enamel; eo,enamel organ; m, dental mesenchyme; od, odontoblast; p, pulp; pd,pre-dentin. Scale bars: 100 μm FIG. 6A-6C, 100 μm FIG. 6D, 25 μm FIG.6E-6F.

FIG. 7. A schematic representation of a healthy tooth (top) and a toothin which the pulp is exposed (bottom).

FIG. 8A-8F. FIG. 8A A non-penetrating cavity preparation, simulatingthat seen in humans, that cuts through the tubular dentin (yellow) butdoes not penetrate to the pulp cavity (pink). FIG. 8B An adjacentsection to FIG. 8A, stained to identify cells expressing GFP. Thesecavity preparations were generated in transgenic mice that, in thepresence of tamoxifen, undergo a recombination event where Wntresponsive cells express GFP. Pulp cells are Wnt responsive, and in thisanimal, tamoxifen was delivered via liposomal particles identical tothose used to delivery WNT3A protein. The GFP positive cells thereforerepresent Wnt responsive cells in the pulp cavity whose only means oftamoxifen exposure was via the dentinal tubules. FIG. 8B(i) and FIG.8B(ii) show higher magnification of the Wnt-responsive odontoblasts thatexpress GFP as a consequence of liposomal tamoxifen delivery. FIG. 8C-8DThe pulp response to the cavity preparation (that cuts through thetubular dentin but does not penetrate to the pulp cavity) and topicalliposomal PBS delivery. A small amount of reparative dentin is generated(dotted white line in FIG. 8D), in keeping with the body's naturalability to stimulate a repair response. FIG. 8E-8F The pulp response toa similar cavity preparation as in panels FIG. 8C-8D, and topicalL-WNT3A delivery to exposed dentin. Significantly more reparative dentinis observed in the pulp chamber.

DETAILED DESCRIPTION

Methods and compositions are provided for enhancing dentin production,and for delivering a lipophilic agent to pulp tissue of a tooth of anindividual. In some cases, a subject method includes a step ofadministering to the pulp of a tooth of an individual, a Wnt stimulatingcomposition that includes a Wnt stimulator agent, at a dose sufficientto enhance the production of dentin by the pulp. In some cases, asubject method includes a step of contacting exposed dentin of a toothwith a composition that includes a lipophilic agent inserted in thenon-aqueous phase of a lipid structure (e.g., whereby the lipophilicagent penetrates the dentin to the underlying pulp tissue). Kits arealso provided for practicing the methods of the disclosure.

Before the present methods and compositions are described, it is to beunderstood that this invention is not limited to particular method orcomposition described, as such may, of course, vary. It is also to beunderstood that the terminology used herein is for the purpose ofdescribing particular embodiments only, and is not intended to belimiting, since the scope of the present invention will be limited onlyby the appended claims.

Where a range of values is provided, it is understood that eachintervening value, to the tenth of the unit of the lower limit unlessthe context clearly dictates otherwise, between the upper and lowerlimits of that range is also specifically disclosed. Each smaller rangebetween any stated value or intervening value in a stated range and anyother stated or intervening value in that stated range is encompassedwithin the invention. The upper and lower limits of these smaller rangesmay independently be included or excluded in the range, and each rangewhere either, neither or both limits are included in the smaller rangesis also encompassed within the invention, subject to any specificallyexcluded limit in the stated range. Where the stated range includes oneor both of the limits, ranges excluding either or both of those includedlimits are also included in the invention.

Unless defined otherwise, all technical and scientific terms used hereinhave the same meaning as commonly understood by one of ordinary skill inthe art to which this invention belongs. Although any methods andmaterials similar or equivalent to those described herein can be used inthe practice or testing of the present invention, some potential andpreferred methods and materials are now described. All publicationsmentioned herein are incorporated herein by reference to disclose anddescribe the methods and/or materials in connection with which thepublications are cited. It is understood that the present disclosuresupersedes any disclosure of an incorporated publication to the extentthere is a contradiction.

As will be apparent to those of skill in the art upon reading thisdisclosure, each of the individual embodiments described and illustratedherein has discrete components and features which may be readilyseparated from or combined with the features of any of the other severalembodiments without departing from the scope or spirit of the presentinvention. Any recited method can be carried out in the order of eventsrecited or in any other order which is logically possible.

It must be noted that as used herein and in the appended claims, thesingular forms “a”, “an”, and “the” include plural referents unless thecontext clearly dictates otherwise. Thus, for example, reference to “acell” includes a plurality of such cells and reference to “the peptide”includes reference to one or more peptides and equivalents thereof, e.g.polypeptides, known to those skilled in the art, and so forth.

The publications discussed herein are provided solely for theirdisclosure prior to the filing date of the present application. Nothingherein is to be construed as an admission that the present invention isnot entitled to antedate such publication by virtue of prior invention.Further, the dates of publication provided may be different from theactual publication dates which may need to be independently confirmed.

Definitions

In the description that follows, a number of terms conventionally usedin the field are utilized. In order to provide a clear and consistentunderstanding of the specification and claims, and the scope to be givento such terms, the following definitions are provided.

The terms “polypeptide,” “peptide” and “protein” are usedinterchangeably herein to refer to a polymer of amino acid residues. Theterms also apply to amino acid polymers in which one or more amino acidresidue is an artificial chemical mimetic of a corresponding naturallyoccurring amino acid, as well as to naturally occurring amino acidpolymers and non-naturally occurring amino acid polymer.

The term “amino acid” refers to naturally occurring and synthetic aminoacids, as well as amino acid analogs and amino acid mimetics thatfunction in a manner similar to the naturally occurring amino acids.Naturally occurring amino acids are those encoded by the genetic code,as well as those amino acids that are later modified, e.g.,hydroxyproline, gamma-carboxyglutamate, and O-phosphoserine. Amino acidanalogs refers to compounds that have the same basic chemical structureas a naturally occurring amino acid, i.e., an .alpha. carbon that isbound to a hydrogen, a carboxyl group, an amino group, and an R group,e.g., homoserine, norleucine, methionine sulfoxide, methionine methylsulfonium. Such analogs have modified R groups (e.g., norleucine) ormodified peptide backbones, but retain the same basic chemical structureas a naturally occurring amino acid. Amino acid mimetics refers tochemical compounds that have a structure that is different from thegeneral chemical structure of an amino acid, but that functions in amanner similar to a naturally occurring amino acid.

The terms “recipient”, “individual”, “subject”, “host”, and “patient”,are used interchangeably herein and refer to any individual for whomdiagnosis, treatment, or therapy is desired, particularly humans.“Mammal” or “mammalian” for purposes of treatment refers to any animalclassified as a mammal, including humans, domestic and farm animals, andzoo, sports, or pet animals, such as dogs, horses, cats, cows, sheep,goats, pigs, etc. In some embodiments, the mammal is human.

Compositions

The present disclosure provides compositions and methods for enhancingdentin production by dental pulp tissue. Such methods includeadministering to the pulp of a tooth of an individual, a Wnt stimulatingcomposition comprising a Wnt stimulator agent, at a dose sufficient toenhance the production of dentin by the pulp. In some cases the Wntstimulator agent comprises a lipophilic Wnt stimulator agent (e.g., aWnt protein, such as Wnt3A) inserted in the non-aqueous phase of a lipidstructure.

The present disclosure also provides compositions and methods fordelivering a lipophilic agent to pulp tissue of a tooth of anindividual. In some cases the lipophilic agent is a growth factor (e.g.,a growth factor having a lipid moiety). In some cases, the lipophilicagent is a lipophilic Wnt stimulator agent (e.g., a Wnt protein, such asWnt3A) inserted in the non-aqueous phase of a lipid structure.

Lipid Structure.

In some embodiments, the subject agent (e.g., an agent of interest) is alipophilic agent inserted in the non-aqueous phase of a lipid structure.Lipid structures can be important for maintaining the activity oflipophilic agents (e.g., Wnt proteins, growth factors, etc., e.g.,having a lipid moiety), following in vivo administration. The subjectlipophilic agents (e.g., Wnt proteins, growth factors, etc., e.g.,having a lipid moiety) are not encapsulated in the aqueous phase of thelipid structures, but are rather integrated into the lipid membrane, andmay be inserted in the outer layer of a membrane. Such a structure isnot predicted from conventional methods of formulating agents (e.g.,proteins) in, for example, liposomes. A Wnt protein integrated withinsuch lipid structure is referred herein as L-Wnt (e.g., Wnt3A integratedinto such a lipid structure can be referred to as L-Wnt3A). The methodsused for tethering lipophilic agents (e.g., Wnt proteins) to theexternal surface of a liposome or micelle can utilize a moiety (e.g., aprotein might have an amino acid sequence) so as to emphasize theexoliposomal display of the protein. In some cases, crude liposomes arefirst pre-formed and a lipophilic agent (e.g., a Wnt protein, a growthfactors, etc., e.g., having a lipid moiety) can then be added to thecrude mixture, which will favor addition of exo-liposomal agent (e.g.,Wnt protein), followed by various formulation steps, which may includesize filtering; dialysis, and the like. Suitable lipids include fattyacids, neutral fats such as triacylglycerols, fatty acid esters andsoaps, long chain (fatty) alcohols and waxes, sphingoids and other longchain bases, glycolipids, sphingolipids, carotenes, polyprenols,sterols, and the like, as well as terpenes and isoprenoids. For example,molecules such as diacetylene phospholipids may find use. Included arecationic molecules, including lipids, synthetic lipids and lipidanalogs, having hydrophobic and hydrophilic moieties, a net positivecharge, and which by itself can form spontaneously into bilayer vesiclesor micelles in water. Liposomes manufactured with a neutral charge, e.g.DMPC, can be used. Any amphipathic molecules that can be stablyincorporated into lipid micelle or bilayers in combination withphospholipids can be used, with its hydrophobic moiety in contact withthe interior, hydrophobic region of the micelle or bilayer membrane, andits polar head group moiety oriented toward the exterior, polar surfaceof the membrane.

The term “cationic amphipathic molecules” is intended to encompassmolecules that are positively charged at physiological pH, and moreparticularly, constitutively positively charged molecules, comprising,for example, a quaternary ammonium salt moiety. Cationic amphipathicmolecules typically consist of a hydrophilic polar head group andlipophilic aliphatic chains. Similarly, cholesterol derivatives having acationic polar head group may also be useful. See, for example, Farhoodet al. (1992) Biochim. Biophys. Acta 1111:239-246; Vigneron et al.(1996) Proc. Natl. Acad. Sci. (USA) 93:9682-9686. Cationic amphipathicmolecules of interest include, for example, imidazolinium derivatives(WO 95/14380), guanidine derivatives (WO 95/14381), phosphatidyl cholinederivatives (WO 95/35301), and piperazine derivatives (WO 95/14651).Examples of cationic lipids that may be used in the present inventioninclude DOTIM (also called BODAI) (Saladin et al., (1995) Biochem. 34:13537-13544), DDAB (Rose et al., (1991) BioTechniques 10(4):520-525),DOTMA (U.S. Pat. No. 5,550,289), DOTAP (Eibl and Wooley (1979) Biophys.Chern. 10:261-271), DMRIE (Feigner et al., (1994) J. Bioi. Chern.269(4): 2550-2561), EDMPC (commercially available from Avanti PolarLipids, Alabaster, Ala.), DCC hoi (Gau and Huang (1991) Biochem.Biophys. Res. Comm. 179:280-285), DOGS (Behr et al., (1989) Proc. Nat!.Acad. Sci. USA, 86:6982-6986), MBOP (also called MeBOP) (WO 95/14651),and those described in WO 97/00241. While not required for activity, insome embodiments a lipid structure may include a targeting group, e.g. atargeting moiety covalently or non-covalently bound to the hydrophilichead group. Head groups useful to bind to targeting moieties include,for example, biotin, amines, cyano, carboxylic acids, isothiocyanates,thiols, disulfides, ahalocarbonyl compounds, a,p-unsaturated carbonylcompounds, alkyl hydrazines, etc. Chemical groups that find use inlinking a targeting moiety to an amphipathic molecule also includecarbamate; amide (amine plus carboxylic acid); ester (alcohol pluscarboxylic acid), thioether (haloalkane plus sulfhydryl; maleimide plussulfhydryl), Schiffs base (amine plus aldehyde), urea (amine plusisocyanate), thiourea (amine plus isothiocyanate), sulfonamide (amineplus sulfonyl chloride), disulfide; hyrodrazone, lipids, and the like,as known in the art. For example, targeting molecules may be formed byconverting a commercially available lipid, such as DAGPE, a PEG-PDAamine, DOTAP, etc. into an isocyanate, followed by treatment withtriethylene glycol diamine spacer to produce the amine terminatedthiocarbamate lipid which by treatment with the para-isothiocyanophenylglycoside of the targeting moiety produces the desired targetingglycolipids. This synthesis provides a water soluble flexible linkermolecule spaced between the amphipathic molecule that is integrated intothe nanoparticle, and the ligand that binds to cell surface receptors,allowing the ligand to be readily accessible to the protein receptors onthe cell surfaces. Further information about liposomal Wnt compositionsand their use is found in; U.S. patent publication numbers: 20140371151,20120115788, and 20080226707; and U.S. Pat. No. 8,809,272; all of whichare hereby incorporated by reference in their entirety.

In some cases, liposomes or micelles are used as a delivery vehicle. Aliposome is a spherical vesicle with a membrane composed of aphospholipid bilayer. Liposomes can be composed of naturally-derivedphospholipids with mixed lipid chains (like eggphosphatidylethanolamine), or of pure surfactant components like DOPE(dioleolylphosphatidylethanolamine). Liposomes often contain a core ofencapsulated aqueous solution; while lipid spheres that contain noaqueous material are referred to as micelles. As wnt proteins arepresent in the lipid phase and not the encapsulated aqueous phase,micelles may be used interchangeably with liposome for the compositionsof the present disclosure. The lipids may be any useful combination ofknown liposome or micelle forming lipids, including cationic lipids,such as phosphatidylcholine, or neutral lipids, such as cholesterol,phosphatidyl serine, phosphatidyl glycerol, and the like.

In some embodiments, the vesicle-forming lipid is selected to achieve aspecified degree of fluidity or rigidity, to control the stability ofthe structure in serum, etc. Liposomes having a more rigid lipidbilayer, or a liquid crystalline bilayer, are achieved by incorporationof a relatively rigid lipid, e.g., a lipid having a relatively highphase transition temperature, e.g., up to 60° C. Rigid, i.e., saturated,lipids contribute to greater membrane rigidity in the lipid bilayer.Other lipid components, such as cholesterol, are also known tocontribute to membrane rigidity in lipid bilayer structures. Lipidfluidity is achieved by incorporation of a relatively fluid lipid,typically one having a lipid phase with a relatively low liquid toliquid-crystalline phase transition temperature, e.g., at or below roomtemperature.

The liposomes may be prepared by a variety of techniques, such as thosedetailed in Szoka, F., Jr., et al., Ann. Rev. Biophys. Bioeng. 9:467(1980). Typically, the liposomes are multilamellar vesicles (MLVs),which can be formed by simple lipid-film hydration techniques. In thisprocedure, a mixture of liposome-forming lipids of the type detailedabove dissolved in a suitable organic solvent is evaporated in a vesselto form a thin film, which is then covered by an aqueous medium. Thelipid film hydrates to form MLVs, e.g., in some cases with sizes in arange of from 0.1 to 10 microns.

The liposomes, micelles, etc. of the disclosure may have substantiallyhomogeneous sizes in a selected size range, for example, between 0.005to 0.5 microns (e.g., 0.01 to 0.5 0.02 to 0.5, 0.025 to 0.5, 0.05 to0.5, 0.075 to 0.5, 0.1 to 0.5, 0.005 to 0.4, 0.01 to 0.4 0.02 to 0.4,0.025 to 0.4, 0.05 to 0.4, 0.075 to 0.4, 0.1 to 0.4, 0.005 to 0.3, 0.01to 0.3 0.02 to 0.3, 0.025 to 0.3, 0.05 to 0.3, 0.075 to 0.3, 0.1 to 0.3,0.005 to 0.2, 0.01 to 0.2 0.02 to 0.2, 0.025 to 0.2, 0.05 to 0.2, 0.075to 0.2, 0.1 to 0.2, 0.005 to 0.1, 0.01 to 0.1 0.02 to 0.1, 0.025 to 0.1,0.05 to 0.1, 0.075 to 0.1, 0.02 to 0.05, or 0.02 to 0.35 microns).

One effective sizing method for REVs and MLVs involves extruding anaqueous suspension of the liposomes through a series of polycarbonatemembranes having a selected uniform pore size in the range of 0.03 to0.2 micron, typically 0.05, 0.08, 0.1, or 0.2 microns. The pore size ofthe membrane corresponds roughly to the largest sizes of liposomesproduced by extrusion through that membrane, particularly where thepreparation is extruded two or more times through the same membrane.Homogenization methods are also useful for down-sizing liposomes tosizes of 100 nm or less.

The pharmaceutical compositions of the present disclosure can alsocomprise a pharmaceutically acceptable carrier. Many pharmaceuticallyacceptable carriers may be employed in the compositions of the presentdisclosure. Generally, normal saline will be employed as thepharmaceutically acceptable carrier. Other suitable carriers include,e.g., water, buffered water, 0.4% saline, 0.3% glycine, and the like,including glycoproteins for enhanced stability, such as albumin,lipoprotein, globulin, etc. These compositions may be sterilized byconventional, well known sterilization techniques. The resulting aqueoussolutions may be packaged for use or filtered under aseptic conditionsand lyophilized, the lyophilized preparation being combined with asterile aqueous solution prior to administration. The compositions maycontain pharmaceutically acceptable auxiliary substances as required toapproximate physiological conditions, such as pH adjusting and bufferingagents, tonicity adjusting agents and the like, for example, sodiumacetate, sodium lactate, sodium chloride, potassium chloride, calciumchloride, etc.

The concentration of lipid structures in the carrier may vary.Generally, the concentration can be about 0.1 to 1000 mg/ml, usuallyabout 1-500 mg/ml, about 5 to 100 mg/ml, etc. Persons of skill may varythese concentrations to optimize treatment with different lipidcomponents or of particular patients.

Subject compositions can include a therapeutically effective in vivodose of a lipophilic agent (e.g., a wnt protein), and may comprise acocktail of one or more lipophilic agents (e.g., one or more wntproteins, one or more Wnt proteins in addition to one or more otherlipophilic agents, etc.).

Wnt Signaling Pathway/Wnt Proteins

In some embodiments, the subject agent (e.g., an agent of interest) is aWnt stimulator agent. A Wnt stimulator agent increases activity of theWnt signaling pathway in a target cell. A target cell (and/or tissue)that is “Wnt responsive” is a cell/tissue that can respond to theextracellular presence of a Wnt protein by triggering the Wnt signalingpathway. A Wnt responsive cell includes components of the Wnt signalingpathway (described in more detail below), including a receptor (e.g., aFrizzled receptor) that can bind to Wnt proteins. Not all cells are Wntresponsive. In some embodiments, the target cell/tissue is Wntresponsive. In some embodiments the target cell is not Wnt responsive.In some embodiments, it is unknown whether the target cell is Wntresponsive. In some embodiments, it is known whether the target cell isWnt responsive. In some embodiments, the target cell is part of aheterogeneous population of target cells (i.e., a heterogeneous targetcell population) in which some cells are Wnt responsive and some cellsare not Wnt responsive (e.g., in some cases a target tissue, such asdental pulp, includes cells that are Wnt responsive as well as cellsthat are not Wnt responsive). In some embodiments, it is known whichcells of a heterogeneous target cell population are Wnt responsive(e.g., stem cells). In some embodiments, it is unknown which cells of aheterogeneous target cell population are Wnt responsive.

The misregulation of Wnt signaling components at various stages duringembryogenesis leads to catastrophic developmental defects whilemisregulation in adults leads to various disease states, includingcancer. There are two main branches of the Wnt signaling pathway: (1)the canonical β-Catenin dependent Wnt signaling pathway and (2) thenon-canonical β-Catenin independent pathways, which include planar cellpolarity (PCP) signaling as well as Calcium signaling (Gao, et. al, CellSignal. 2010 May; 22(5):717-27. Epub 2009 Dec. 13). As used herein, theterms “Wnt signaling” and “Wnt/β-Catenin signaling” are usedinterchangeably to refer to the canonical β-Catenin dependent Wntsignaling pathway. As such, a Wnt signaling stimulator, also referred toas a “Wnt stimulator agent” (i.e., agonist) (e.g., Wnt3A) increasesoutput from the β-Catenin dependent Wnt signaling pathway while a Wntsignaling inhibitor (i.e., antagonist) decreases output from theβ-Catenin dependent Wnt signaling pathway.

Activation of the Wnt pathway culminates when the protein β-Cateninenters the cell nucleus (for recent review of the canonical β-Catenindependent Wnt signaling pathway see Clevers et. al., Cell. 2012 Jun. 8;149(6):1192-205: Wnt/β-catenin signaling and disease). However, in theabsence of Wnt signaling, free cytosolic β-Catenin is incorporated intoa complex, known in the art as the β-Catenin destruction complex, whichincludes the proteins Axin, Adenomatous Polyposis Coli (APC), andglycogen synthase kinase (GSK-3β). Phosphorylation of β-Catenin byGSK-3β designates β-Catenin for the ubiquitin pathway and degradation(e.g., via βIRCP).

Transduction of the β-Catenin dependent Wnt signaling pathway (i.e., theWnt signaling pathway) is triggered by the binding of secreted Wntligands to two distinct families of cell-surface receptors: the Frizzled(Fz) receptor family and the LDL-receptor-related protein (LRP) family(Akiyama, Cytokine Growth Factor Rev. 11:273-82 (2000)). This bindingleads to the activation of Dishevelled (Dvl) proteins, which inhibitglycogen synthase kinase-3β (GSK-3β) activity (i.e., phosphorylation ofβ-Catenin), leading to the cytosolic stabilization of β-Catenin.Stabilized β-Catenin then enters the nucleus and associates with theTCF/LEF (T Cell-specific transcription Factor/Lymphoid Enhancer Factor)family of transcription factors to induce transcription of downstreamtarget genes.

In the absence of Wnt signaling, cytosolic (and therefore nuclear)levels of β-Catenin are kept low by negative regulatory components ofthe pathway while in the presence of Wnt signaling, cytosolic (andtherefore nuclear) levels of β-Catenin are stabilized by positiveregulatory components of the pathway. For this reason, β-Catenin levels(e.g., monitored via Western blot) can provide insight into whether theWnt signaling pathway of a cell has been stimulated or inhibited (e.g.,increased levels of β-Catenin indicate increased signaling and decreasedlevels indicate decrease signaling). Likewise, β-Catenin levels in thenucleus (e.g., monitored via fluorescence microscopy, Western blot,etc.) can also be monitored to determine increased or decreasedsignaling.

By “positive regulatory components” of the Wnt pathway, it is meantproteins that function by enhancing (i.e., stimulating) the Wnt pathway,thus resulting in increased Wnt pathway signaling activity (i.e.,increased Wnt pathway signaling output, e.g., increased target geneexpression, increased reporter activity, increased levels of β-Catenin,etc.). Examples of known positive regulatory components of the Wntpathway include, but are in no way limited to: Wnt (secreted,extracellular), Norrin (secreted, extracellular), R-spondin (secreted,extracellular), PORCN, WIs, Frizzled, LRP5 and LRP6, Tspan12, Lgr4,Lgr5, Lgr6, Dvl, β-Catenin, and TCF/LEF. A secreted positive regulatorycomponent of the Wnt pathway (e.g., Wnt, Norrin, R-spondin, and thelike) is referred to herein as a “Wnt stimulator polypeptide”. In somecases a Wnt stimulator polypeptide is a Wnt protein.

Suitable Wnt polypeptides (i.e., Wnt proteins) include, but are in noway limited to human Wnt polypeptides. Human Wnt proteins of interest inthe present application include the following (accession numbers are formRNAs encoding the associated Wnt protein): Wnt-1 (GenBank Accession No.NM_005430); Wnt-2 (GenBank Accession No. NM_003391); Wnt-2B (Wnt-13)(GenBank Accession No. NM_004185 (isoform 1), NM_024494.2 (isoform 2)),Wnt-3 (RefSeq.: NM_030753), Wnt3a (GenBank Accession No. NM_033131),Wnt-4 (GenBank Accession No. NM_030761), Wnt-5A (GenBank Accession No.NM_003392), Wnt-5B (GenBank Accession No. NM_032642), Wnt-6 (GenBankAccession No. NM_006522), Wnt-7A (GenBank Accession No. NM_004625),Wnt-7B (GenBank Accession No. NM_058238), Wnt-8A (GenBank Accession No.NM_058244), Wnt-8B (GenBank Accession No. NM_003393), Wnt-9A (Wnt-14)(GenBank Accession No. NM_003395), Wnt-9B (Wnt-15) (GenBank AccessionNo. NM_003396), Wnt-10A (GenBank Accession No. NM_025216), Wnt-10B(GenBank Accession No. NM_003394), Wnt-11 (GenBank Accession No.NM_004626), Wnt-16 (GenBank Accession No. NM_016087)). Although eachmember has varying degrees of sequence identity with the family, allencode small (i.e., 39-46 kD), acylated, palmitoylated, secretedglycoproteins that contain 23-24 conserved cysteine residues whosespacing is highly conserved (McMahon, A P et al., Trends Genet. 1992; 8:236-242; Miller, J R. Genome Biol. 2002; 3(1): 3001.1-3001.15). OtherWnt polypeptides of interest in the present invention include orthologsof the above from any mammal, including domestic and farm animals, andzoo, laboratory or pet animals, dogs, cats, cattle, horses, sheep, pigs,goats, rabbits, rats, mice, frogs, zebra fish, fruit fly, worm, etc.

Wnt proteins form a family of highly conserved secreted signalingmolecules that regulate cell-to-cell interactions during embryogenesis.The terms “Wnts” or “Wnt gene product” or “Wnt protein” or “Wntpolypeptide” are used interchangeable and encompass native sequence Wntpolypeptides, Wnt polypeptide variants, Wnt polypeptide fragments andchimeric Wnt polypeptides. In some embodiments of the invention, the Wntprotein comprises palmitate covalently bound to a cysteine residue. A“native sequence” polypeptide is one that has the same amino acidsequence as a Wnt polypeptide derived from nature, regardless of themethod used for its production. Such native sequence polypeptides can beisolated from cells producing endogenous Wnt protein or can be producedby recombinant or synthetic means. Thus, a native sequence polypeptidecan have the amino acid sequence of, e.g. naturally occurring humanpolypeptide, murine polypeptide, or polypeptide from any other mammalianspecies, or from non-mammalian species, e.g. Drosophila, C. elegans, andthe like.

The term “native sequence Wnt polypeptide” includes, without limitation,human and murine Wnt polypeptides. Human Wnt proteins include thefollowing: Wnt1, Genbank reference NP005421.1; Wnt2, Genbank referenceNP003382.1, which is expressed in brain in the thalamus, in fetal andadult lung and in placenta; two isoforms of Wnt2B, Genbank referencesNP004176.2 and NP078613.1. Isoform 1 is expressed in adult heart, brain,placenta, lung, prostate, testis, ovary, small intestine and colon. Inthe adult brain, it is mainly found in the caudate nucleus, subthalamicnucleus and thalamus. Also detected in fetal brain, lung and kidney.Isoform 2 is expressed in fetal brain, fetal lung, fetal kidney, caudatenucleus, testis and cancer cell lines. Wnt 3 and Wnt3A play distinctroles in cell-cell signaling during morphogenesis of the developingneural tube, and have the Genbank references NP11 0380.1 and X56842(Swiss-Prot P56704), respectively.

The native human Wnt3A amino acid sequence (NP_149122.1) is specificallydisclosed as SEQ ID NO: 19. Wnt 4 has the Genbank reference NP11 0388.2.Wnt 5A and Wnt 5B have the Genbank references NP003383.1 and AK013218.Wnt 6 has the Genbank reference NP006513.1; Wnt 7A has the Genbankreference NP004616.2. Wnt 7B has the Genbank reference NP478679.1. Wnt8A has two alternative transcripts, Genbank references NP114139.1 andNP490645.1. Wnt 8B has the Genbank reference NP003384.1. Wnt 10A has theGenbank reference NP079492.2. Wnt 10B has the Genbank referenceNP003385.2. Wnt 11 has the Genbank reference NP004617.2. Wnt 14 has theGenbank reference NP003386.1. Wnt 15 has the Genbank referenceNP003387.1. Wnt 16 has two isoforms, Wnt-16a and Wnt-16b, produced byalternative splicing, Genbank references are NP057171.2 and NP476509.1.All GenBank, SwissProt and other database sequences listed are expresslyincorporated by reference herein.

The term “native sequence Wnt protein” or “native sequence Wntpolypeptide” includes the native proteins with or without the initiatingN-terminal methionine (Met), and with or without the native signalsequence. The terms specifically include the 352 amino acids long nativehuman Wnt3a polypeptide, without or without its N terminal methionine(Met), and with or without the native signal sequence.

A “variant” polypeptide means a biologically active polypeptide asdefined below having less than 100% sequence identity with a nativesequence polypeptide. Such variants include polypeptides wherein one ormore amino acid residues are added at the N- or C-terminus of, orwithin, the native sequence; from about one to forty amino acid residuesare deleted, and optionally substituted by one or more amino acidresidues; and derivatives of the above polypeptides, wherein an aminoacid residue has been covalently modified so that the resulting producthas a non-naturally occurring amino acid. Ordinarily, a biologicallyactive Wnt variant will have an amino acid sequence having at leastabout 90% amino acid sequence identity with a native sequence Wntpolypeptide, preferably at least about 95%, more preferably at leastabout 99%.

A “chimeric” Wnt polypeptide is a polypeptide comprising a Wntpolypeptide or portion (e.g., one or more domains) thereof fused orbonded to heterologous polypeptide. The chimeric Wnt polypeptide willgenerally share at least one biological property in common with a nativesequence Wnt polypeptide. Examples of chimeric polypeptides includeimmunoadhesins, combine a portion of the Wnt polypeptide with animmunoglobulin sequence, and epitope tagged polypeptides, which comprisea Wnt polypeptide or portion thereof fused to a “tag polypeptide”. Thetag polypeptide has enough residues to provide an epitope against whichan antibody can be made, yet is short enough such that it does notinterfere with biological activity of the Wnt polypeptide. Suitable tagpolypeptides generally have at least six amino acid residues and usuallybetween about 6-60 amino acid residues.

A “functional derivative” of a native sequence Wnt polypeptide is acompound having a qualitative biological property in common with anative sequence Wnt polypeptide. “Functional derivatives” include, butare not limited to, fragments of a native sequence and derivatives of anative sequence Wnt polypeptide and its fragments, provided that theyhave a biological activity in common with a corresponding nativesequence Wnt polypeptide. The term “derivative” encompasses both aminoacid sequence variants of Wnt polypeptide and covalent modificationsthereof.

One may determine the specific activity of a Wnt protein in acomposition by determining the level of activity in a functional assay,for example in an in vitro assay, or after in vivo administration in atest model, e.g. accelerating bone regeneration, upregulation of stemcell proliferation, etc., quantitating the amount of Wnt protein presentin a non-functional assay, e.g. immunostaining, ELISA, quantitation onCoomasie or silver stained gel, etc., and determining the ratio of invivo biologically active Wnt to total Wnt.

The effective dose of the Wnt protein may vary depending on the source,purity, preparation method, etc. Where the Wnt protein is L-Wnt3A, theeffective dose is usually at least 0.1 μg/ml, at least 0.5 μg/ml, atleast 1 μg/ml, at least 2.5 μg/ml, at least 5 μg/ml, at least 7.5 μg/ml,at least 10 μg/ml, at least 15 μg/ml, and may be at least 25 μg/ml, atleast 50 μg/ml, or at least 100 μg/ml.

As discussed above, in some embodiments, a Wnt protein (e.g., Wnt3A,e.g., human Wnt3A) is inserted in the non-aqueous phase of a lipidstructure, e.g. in the surface of a liposome, micelle, lipid raft, etc.,in an emulsion, and the like. In some embodiments the Wnt protein ispresented in its active conformation on an outer liposome membrane ormicelle. Where the lipid structure is a liposome it can be desirablethat the Wnt protein not be encapsulated within the liposome, e.g. in anaqueous phase. The lipid-containing particles typically display the Wntprotein, the particles comprising at least one copy of a wnt proteinbearing at least one lipid moiety, where the composition contains atleast 50% of the Wnt polypeptides displayed on the exterior surface ofthe particle. In some cases, R-spondin can be included in the aqueouscore of a liposomal WNT3A (L-Wnt3A) and in so doing, amplify and extendthe Wnt dependent activation of pulp cells.

For example, see Dhamdhere et al., PLoS One. 2014 Jan. 6; 9(1):e83650;and Zhao et al., Methods Enzymol. 2009; 465:331-47, both of which arehereby incorporated by reference in their entirety.

A subject Wnt stimulator agent is any molecule (e.g., a chemicalcompound; a non-coding nucleic acid, e.g., a non-coding RNA; apolypeptide; a nucleic acid encoding a polypeptide, etc.) that resultsin increased output (i.e., increased target gene expression) from theWnt signaling pathway. For example, a Wnt stimulator agent can functionby stabilizing, enhancing the expression of, or enhancing the functionof a positive regulatory component of the pathway or by destabilizing,decreasing the expression of, or inhibiting the function of a negativeregulatory component of the pathway. Thus, a Wnt stimulator agent can bea positive regulatory component of the pathway (e.g., a Wnt protein), ora nucleic acid encoding one or more positive regulatory components ofthe pathway. A Wnt stimulator agent can also be a small molecule ornucleic acid that stabilizes a positive regulatory component of thepathway either at the level of mRNA or protein.

In some embodiments, a Wnt stimulator agent functions by stabilizingβ-Catenin, thus allowing nuclear levels of β-Catenin to rise. β-Catenincan be stabilized in multiple different ways. As multiple differentnegative regulatory components of the Wnt signaling pathway function byfacilitating the degradation of β-Catenin, a subject Wnt stimulatoragent can be a small molecule or nucleic acid inhibitor (e.g., microRNA,shRNA, etc.)(functioning at the level of mRNA or protein) of a negativeregulatory component of the pathway. For example, in some embodiments,the Wnt stimulator agent is an inhibitor of GSK-3β. In some suchembodiments, the inhibitor of GSK-3β is a small molecule chemicalcompound (e.g., TWS119, BIO, CHIR-99021, SB 216763, SB 415286,CHIR-98014 and the like).

TWS119: 3-(6-(3-aminophenyl)-7H-pyrrolo[2,3-d]pyrimidin-4-yloxy)phenolis described by Ding et. al, Proc Natl Acad Sci USA. 2003 Jun. 24;100(13):7632-7. BIO:6-bromo-3-[(3E)-1,3-dihydro-3-(hydroxyimino)-2H-indol-2-ylidene]-1,3-dihydro-(3Z)-2H-indol-2-oneor (2′Z,3E)-6-Bromoindirubin-3′-oxime is described by Meijer et. al,Chem Biol. 2003 December; 10(12):1255-66. CHIR-99021:6-[[2-[[4-(2,4-dichlorophenyl)-5-(5-methyl-1H-imidazol-2-yl)-2-pyrimidinyl]amino]ethyl]amino]-3-pyridinecarbonitrileis described by Bennett et al., J Biol Chem. 2002 Aug. 23;277(34):30998-1004. SB 216763:3-(2,4-dichlorophenyl)-4-(1-methyl-1H-indol-3-yl)-1H-pyrrole-2,5-dioneis described by Cross et al., J Neurochem. 2001 April; 77(1):94-102. SB415286:3-(3-chloro-4-hydroxyphenylamino)-4-(2-nitrophenyl)-1H-pyrrole-2,5-dioneis described by Cross et al., J Neurochem. 2001 April; 77(1):94-102.CHIR-98014:N2-(2-(4-(2,4-dichlorophenyl)-5-(1H-imidazol-1-yl)pyrimidin-2-ylamino)ethyl)-5nitropyridine-2,6-diamine is described by Ring et al., Diabetes. 2003March; 52(3):588-95. Each reference is herein specifically incorporatedby reference.

In some cases, a Wnt stimulator agent is a lipophilic agent. For thoseagents above that are not lipophilic, a Wnt stimulator agent can be suchan agent conjugated to a lipid moiety.

The effective dose of a Wnt stimulator agent can be at least 0.1 μM, atleast 1 μM, at least 2.5 μM, at least 5 μM, and usually not more than500 μM, not more than 250 μM, not more than 100 μM, or not more than 50μM.

By “negative regulatory components” of the Wnt pathway, it is meantproteins that function by antagonizing (i.e., inhibiting) the Wntpathway, thus resulting in decreased pathway output (i.e., decreased Wntpathway signaling output, e.g., decreased target gene expression,decreased reporter activity, decreased levels of β-Catenin, etc.).Examples of known negative regulatory components of the Wnt pathwayinclude, but are in no way limited to: WIF, sFRP, DKK, Wnt5, Wnt11,Notum, WISE/SOST, Axin, APC, GSK-3β, CK1γ, WTX, and βTrCP. A secretednegative regulatory component of the Wnt pathway is referred to hereinas a “Wnt inhibitor polypeptide”.

Wnt inhibitor polypeptides (i.e., secreted negative regulatorycomponents of the Wnt signaling pathway) include members of the WIF (Wntinhibitory factor), sFRP (Secreted Frizzled Related Protein), DKK(Dickkopf), Notum, and WISE/SOST families, which interfere with theappropriate interactions among Wnt, Frizzled, and LRP proteins(Melkonyan et al., 1997, Proc Natl Acad Sci USA 94(25):13636-41; Moon etal., 1997, Cell 88(6):725-8; Fedi et al., 1999, J Biol Chem274(27):19465-72; Nusse, 2001, Nature 411(6835):255-6; Clevers et. al.,Cell. 2012 Jun. 8; 149(6):1192-205: Wnt/β-catenin signaling anddisease). Although most Wnt polypeptides are Wnt stimulatorpolypeptides, certain Wnt polypeptides (e.g., Wnt5 and Wnt11) are Wntinhibitor polypeptides. Wnt5 and Wnt11 have been demonstrated tostimulate non-canonical (non-β-catenin dependent) Wnt signaling and havealso been demonstrated to inhibit canonical (β-catenin dependent) Wntsignaling. Thus, the term “Wnt polypeptide” encompasses some Wntstimulator polypeptides as well as some Wnt inhibitor polypeptides.

The above described agents can be prepared in a variety of ways. Forexample, a subject Wnt stimulating composition and/or a subjectliphophilic agent (e.g., a Wnt protein) agent can be prepared (togetheror separately): as a dosage unit, with a pharmaceutically acceptableexcipient, with pharmaceutically acceptable salts and esters, etc.Compositions can be provided as pharmaceutical compositions.

Pharmaceutical Compositions

Suitable agents can be provided in pharmaceutical compositions suitablefor therapeutic use, e.g. for human treatment. In some embodiments,pharmaceutical compositions of the present disclosure include one ormore therapeutic entities of the present disclosure (e.g., a subject Wntstimulating composition and/or a subject lipophilic agent inserted inthe non-aqueous phase of a lipid structure) and include apharmaceutically acceptable carrier, a pharmaceutically acceptable salt,a pharmaceutically acceptable excipient, and/or esters or solvatesthereof. In some embodiments, the use of a subject Wnt stimulatingcomposition and/or a subject lipophilic agent inserted in thenon-aqueous phase of a lipid structure includes use in combination withanother therapeutic agent (e.g., a dentin-stimulating agent, a pulpsurvival agent, an anti-infection agent, and the like). Therapeuticformulations that include a subject Wnt stimulating composition and/or asubject lipophilic agent inserted in the non-aqueous phase of a lipidstructure can be prepared by mixing the agent(s) having the desireddegree of purity with a physiologically acceptable carrier, apharmaceutically acceptable salt, an excipient, and/or a stabilizer(Remington's Pharmaceutical Sciences 16th edition, Osol, A. Ed. (1980))(e.g., in the form of lyophilized formulations or aqueous solutions). Acomposition having a subject Wnt stimulating composition and/or asubject lipophilic agent inserted in the non-aqueous phase of a lipidstructure can be formulated, dosed, and administered in a fashionconsistent with good medical practice. Factors for consideration in thiscontext include the particular disorder being treated, the particularmammal being treated, the clinical condition of the individual patient,the cause of the disorder, the site of delivery of the agent, the methodof administration, the scheduling of administration, and other factorsknown to medical practitioners.

“Pharmaceutically acceptable excipient” means an excipient that isuseful in preparing a pharmaceutical composition that is generally safe,non-toxic, and desirable, and includes excipients that are acceptablefor veterinary use as well as for human pharmaceutical use. Suchexcipients can be solid, liquid, semisolid, or, in the case of anaerosol composition, gaseous.

“Pharmaceutically acceptable salts and esters” means salts and estersthat are pharmaceutically acceptable and have the desiredpharmacological properties. Such salts include salts that can be formedwhere acidic protons present in the compounds are capable of reactingwith inorganic or organic bases. Suitable inorganic salts include thoseformed with the alkali metals, e.g. sodium and potassium, magnesium,calcium, and aluminum. Suitable organic salts include those formed withorganic bases such as the amine bases, e.g., ethanolamine,diethanolamine, triethanolamine, tromethamine, N-methylglucamine, andthe like. Such salts also include acid addition salts formed withinorganic acids (e.g., hydrochloric and hydrobromic acids) and organicacids (e.g., acetic acid, citric acid, maleic acid, and the alkane- andarene-sulfonic acids such as methanesulfonic acid and benzenesulfonicacid). Pharmaceutically acceptable esters include esters formed fromcarboxy, sulfonyloxy, and phosphonoxy groups present in the compounds,e.g., C₁₋₆ alkyl esters. When there are two acidic groups present, apharmaceutically acceptable salt or ester can be a mono-acid-mono-saltor ester or a di-salt or ester; and similarly where there are more thantwo acidic groups present, some or all of such groups can be salified oresterified. Compounds named in this invention can be present inunsalified or unesterified form, or in salified and/or esterified form,and the naming of such compounds is intended to include both theoriginal (unsalified and unesterified) compound and its pharmaceuticallyacceptable salts and esters. Also, certain compounds named in thisinvention may be present in more than one stereoisomeric form, and thenaming of such compounds is intended to include all single stereoisomersand all mixtures (whether racemic or otherwise) of such stereoisomers.

The terms “pharmaceutically acceptable”, “physiologically tolerable” andgrammatical variations thereof, as they refer to compositions, carriers,diluents and reagents, are used interchangeably and represent that thematerials are capable of administration to or upon a human without theproduction of undesirable physiological effects to a degree that wouldprohibit administration of the composition.

“Dosage unit” refers to physically discrete units suited as unitarydosages for the particular individual to be treated. Each unit cancontain a predetermined quantity of active compound(s) calculated toproduce the desired therapeutic effect(s) in association with therequired pharmaceutical carrier. The specification for the dosage unitforms can be dictated by (a) the unique characteristics of the activecompound(s) and the particular therapeutic effect(s) to be achieved, and(b) the limitations inherent in the art of compounding such activecompound(s).

In some embodiments, pharmaceutical compositions can include large,slowly metabolized macromolecules such as proteins, polysaccharides suchas chitosan, polylactic acids, polyglycolic acids and copolymers (suchas latex functionalized Sepharose™, agarose, cellulose, and the like),polymeric amino acids, amino acid copolymers, and lipid aggregates (suchas oil droplets or liposomes).

Acceptable carriers, excipients, or stabilizers are non-toxic torecipients at the dosages and concentrations employed, and includebuffers such as phosphate, citrate, and other organic acids;antioxidants including ascorbic acid and methionine; preservatives (suchas octadecyidimethylbenzyl ammonium chloride; hexamethonium chloride;benzalkonium chloride, benzethonium chloride; phenol, butyl or benzylalcohol; alkyl parabens such as methyl or propyl paraben; catechol;resorcinol; cyclohexanol; 3-pentanol; and m-cresol); low molecularweight (less than about 10 residues) polypeptides; proteins, such asserum albumin, gelatin, or immunoglobulins; hydrophilic polymers such aspolyvinylpyrrolidone; amino acids such as glycine, glutamine,asparagine, histidine, arginine, or lysine; monosaccharides,disaccharides, and other carbohydrates including glucose, mannose, ordextrins; chelating agents such as EDTA; sugars such as sucrose,mannitol, trehalose or sorbitol; salt-forming counter-ions such assodium; metal complexes (e.g., Zn-protein complexes); and/or non-ionicsurfactants such as TWEEN™, PLURONICS™ or polyethylene glycol (PEG).Formulations to be used for in vivo administration must be sterile. Thisis readily accomplished by filtration through sterile filtrationmembranes.

Ingredients may also be entrapped in microcapsule prepared, for example,by coacervation techniques or by interfacial polymerization, forexample, hydroxymethylcellulose or gelatin-microcapsule andpoly-(methylmethacylate) microcapsule, respectively, in colloidal drugdelivery systems (for example, liposomes, albumin microspheres,microemulsions, nano-particles and nanocapsules) or in macroemulsions.Such techniques are disclosed in Remington's Pharmaceutical Sciences16th edition, Osol, A. Ed. (1980).

Compositions can be prepared as injectables, either as liquid solutionsor suspensions; solid forms suitable for solution in, or suspension in,liquid vehicles prior to injection can also be prepared. The preparationalso can be emulsified or encapsulated in liposomes or micro particlessuch as polylactide, polyglycolide, or copolymer for enhanced adjuvanteffect, as discussed above. Langer, Science 249: 1527, 1990 and Hanes,Advanced Drug Delivery Reviews 28: 97-119, 1997. The agents of thisinvention can be administered in the form of a depot injection orimplant preparation which can be formulated in such a manner as topermit a sustained or pulsatile release of the active ingredient. Thepharmaceutical compositions can be formulated as sterile, substantiallyisotonic and in full compliance with all Good Manufacturing Practice(GMP) regulations of the U.S. Food and Drug Administration.

Methods

The present disclosure provides compositions and methods for enhancingdentin production by dental pulp tissue. Such methods can includeadministering to the pulp of a tooth of an individual, a Wnt stimulatingcomposition that includes a Wnt stimulator agent, at a dose sufficientto enhance the production of dentin by the pulp. In some cases, the pulpis exposed pulp and said administering includes contacting the exposedpulp with the Wnt stimulating composition. In some cases, saidadministering includes contacting dentin with the Wnt stimulatingcomposition, whereby the Wnt stimulating composition penetrates thedentin to the underlying pulp tissue. As discussed above, the Wntstimulator agent can be a lipophilic Wnt stimulator agent. In somecases, the Wnt stimulator agent is a Wnt protein (e.g., a Wnt proteinhaving a lipid moiety)(e.g., a Wnt3A protein). In some cases, the Wntstimulating composition includes a lipophilic Wnt stimulator agent(e.g., a Wnt protein, e.g., having a lipid moiety; a Wnt3A protein,e.g., having a lipid moiety; and the like) inserted in the non-aqueousphase of a lipid structure. Thus, in some cases, a lipophilic agentinserted in the non-aqueous phase of a lipid structure is an L-Wnt(e.g., L-Wnt3A).

The present disclosure provides compositions and methods for deliveringa lipophilic agent to pulp tissue of a tooth of an individual. Suchmethods can include contacting dentin of the tooth (e.g., exposeddentin) with a composition that includes a lipophilic agent inserted inthe non-aqueous phase of a lipid structure (e.g., a liposomes, micelles,and the like), whereby the lipophilic agent penetrates the dentin to theunderlying pulp tissue. In some cases, the individual has toothsensitivity. In some cases, the pulp tissue of said tooth is notexposed. In some cases, the pulp tissue of said tooth is exposed. Insome cases, the method includes, prior to contacting the dentin, a stepof exposing the dentin. In some cases, the lipophilic agent is a growthfactor (e.g., a growth factor having a lipid moiety). In some cases, thelipophilic agent is a lipophilic Wnt stimulator agent. In some cases,the Wnt stimulator agent is a Wnt protein (e.g., a Wnt protein having alipid moiety)(e.g., a Wnt3A protein). Thus, in some cases, a lipophilicagent inserted in the non-aqueous phase of a lipid structure is an L-Wnt(e.g., L-Wnt3A).

In some cases, the method includes, prior to contacting dentin (e.g.,prior to contacting dentin with a composition having a lipophilic agentinserted in the non-aqueous phase of a lipid structure), a step ofexposing dentin. For example, when practicing a subject method, (e.g.,as part of a dental procedure), existing metal or plastic restorations,carious dentin, or other medicants (e.g., pulp-lining materials) can beremoved, thus exposing dentin. In some cases, exposed dentin is cleaned(e.g, with a mild solvent such as ethylenediaminetetraacetic acid(EDTA), e.g., 15-17%) prior to contacting the dentin, e.g., to remove asmear layer. In some cases, the dentin surface can be rinsed priorcontacting the dentin (e.g., prior to contacting the dentin with acomposition comprising a lipophilic agent inserted in the non-aqueousphase of a lipid structure). In some cases, the dentin surface can berinsed and then gently air-dried prior to contacting dentin (e.g., priorto contacting the dentin with a composition comprising a lipophilicagent inserted in the non-aqueous phase of a lipid structure). In somecases, (e.g., after removal of decayed enamel, and for example if thepreparation extends into the dentin), a subject composition having alipophilic agent inserted in the non-aqueous phase of a lipid structure(e.g, L-Wnt, L-Wnt3A, and the like) may be applied to the dentin (e.g.,the dentin can be contacted with the subject composition) to stimulatepulp cells to produce additional dentin.

In some cases, the individual (e.g., the individual to be treated usingthe subject methods and/or compositions) has tooth sensitivity. When anindividual has sensitive teeth, certain activities, such as brushing,flossing, eating and drinking, can cause sharp, temporary pain in theteeth. Sensitive teeth can be the result of worn tooth enamel or exposedtooth roots. Tooth sensitivity can be caused by factors such as acavity, a carious lesion, a cracked or chipped tooth, a recently placedfilling or a side effect of other dental procedures (e.g., dentalrestoration, bleaching, and the like), periodontal disease, and/or as aresult of aging. In some cases, the pulp of the tooth to be treated isexposed (e.g., via chipping, cavity, a dental procedure, etc.)(e.g., seeFIG. 7). In some cases, the pulp of the tooth to be treated is notexposed. In some cases, the individual (e.g., the individual to betreated using the subject methods and/or compositions) has one or moreof: a cavity, a carious lesion, a cracked or chipped tooth, a recentlyplaced filling, a side effect of a dental procedure (e.g., dentalrestoration, bleaching, and the like), and periodontal disease.

For example, in some cases, a carious lesion may appear (e.g.,radiographically appear) to be near or to impinge upon the pulp cavity.The subject methods (e.g., the application of L-WNT3A) can be used toactivate stem cells, progenitor cells, and/or odontoblasts within theviable pulp cavity and in doing so stimulate/enhance dentin formation.The dentin can act as an insulator and can protect the remaining pulptissue (e.g., from trauma).

In some cases, pulp tissue of a tooth is exposed and the method is amethod of administering to the pulp of the tooth of an individual, a Wntstimulating composition that includes a Wnt stimulator agent, at a dosesufficient to enhance the production of dentin by the pulp. In somecases, the pulp tissue can be exposed due to injury, a carious lesion,etc. (e.g., a chipped tooth, a cavity, and the like). In some cases, thepulp tissue can be exposed intentionally (e.g., by a person performing adental procedure). For example, the pulp tissue can be exposed during apreparation of the tooth for the subject methods, and/or for some otherdental procedure. In some cases, a subject method includes a step ofexposing pulp of a tooth to produce the exposed pulp. In some cases, theadministering includes contacting the exposed pulp with a Wntstimulating composition, which includes a Wnt stimulator agent. In somecases, the Wnt stimulator agent is a lipophilic Wnt stimulator agent. Insome cases, the Wnt stimulator agent is a Wnt protein (e.g., a Wntprotein having a lipid moiety)(e.g., a Wnt3A protein, e.g., having alipid moiety). In some cases, the Wnt stimulating composition includes alipophilic Wnt stimulator agent (e.g., a Wnt protein, e.g., having alipid moiety; a Wnt3A protein, e.g., having a lipid moiety; and thelike) inserted in the non-aqueous phase of a lipid structure. Thus, insome cases, a Wnt stimulating composition includes L-Wnt (e.g.,L-Wnt3A). In some cases, the pulp tissue can be exposed unintentionally(e.g., by a person performing a dental procedure).

The terms “treatment”, “treating”, “treat” and the like are used hereinto generally refer to obtaining a desired pharmacologic and/orphysiologic effect. The effect can be prophylactic in terms ofcompletely or partially preventing a disease or symptom(s) thereofand/or may be therapeutic in terms of a partial or completestabilization or cure for a disease and/or adverse effect attributableto the disease. The subject methods are useful for both prophylactic andtherapeutic purposes. Thus, as used herein, the term “treating” is usedto refer to both treatment of a pre-existing condition (e.g., toothsensitivity, pulp exposure, and the like), and to preventativetreatments (e.g., to increase dentin of a tooth prior to symptomsassociated with reduced dentin; e.g., as a way to prevent, reduce thelikelihood of, or reduce the severity of future tooth sensitivityfollowing a dental procedure). Evidence of therapeutic effect may be anydiminution in the severity of the condition relative to the pre-existingcondition or relative to the expected outcome in the absence oftreatment. The therapeutic effect can be measured in terms of clinicaloutcome or can be determined by immunological or biochemical tests(e.g., tests to determine if Wnt signaling activity was induced).Individuals to be treated can be mammals, e.g. primates, includinghumans, may be laboratory animals, e.g. rabbits, rats, mice, etc.,particularly for evaluation of therapies, horses, dogs, cats, farmanimals, etc.

A therapeutic treatment is one in which the subject is inflicted priorto administration and a prophylactic treatment is one in which thesubject is not inflicted prior to administration. In some embodiments,the subject has an increased likelihood of becoming inflicted or issuspected of being inflicted prior to treatment. In some embodiments,the subject is suspected of having an increased likelihood of becominginflicted.

As used herein, the term “pulp exposure” or “dental pulp exposure”refers to exposure of the dental pulp tissue. In healthy teeth, pulpresides within the dentin (e.g., see FIG. 7), but pulp exposure can leadto inflammation, infection, abscess formation, etc. Pulp exposure canresult from traumatic injury (e.g., a cracked tooth), decay, cavityformation, and the like. In some cases, pulp is exposed during (e.g., asa result of) a dental procedure (which may be intentional orunintentional exposure). Pulp exposure can be acute (e.g., a crackedtooth), or can occur over a period of time (e.g., via formation of acavity). In some cases, a subject method includes a step of exposingpulp of a tooth prior to contacting the pulp with a subject Wntstimulatory composition.

In some cases, the subject methods are performed after placement of adeep restoration (amalgam, composite resin, crown) that radiographicallyappears to be near to, or impinges upon, the pulp cavity. In some cases,the subject methods are performed after direct pulp capping in youngpatients, or in patients in which the pulp cavity is inadvertentlyexposed. In some cases, the subject methods are performed afterplacement of any tooth restoration (amalgam, composite resin, crown), toprevent and/or to reduce the likelihood of tooth sensitivity. In somecases, a subject Wnt stimulating composition is topically applied toroot surfaces after root planning and scaling for periodontal diseasepatients. In some cases, a subject Wnt stimulating composition (e.g.,L-Wnt, L-Wnt3A, and the like) is applied to teeth with incipient cariouslesions for which removal of the decayed enamel is generally consideredsufficient. All of the methods described herein have broad applicationsin general restorative dentistry, prosthodontics, and periodontics.

In some cases, the subject methods are a treatment of sensitive teeth(tooth sensitivity), e.g., caused by a dental restoration, a cariouslesion, periodontal disease, or as a result of aging. For example, inteeth where a carious lesion radiographically appears to be near to orimpinges upon the pulp cavity, the application of a subject composition(e.g., L-WNT3A) can be used to activate stem cells, progenitor cells,and/or odontoblasts within the viable pulp cavity and in doing sostimulate tertiary dentin formation. This tertiary dentin can act as aninsulator and protects the remaining pulp tissue from trauma.

In some cases, a Wnt stimulating composition and/or a lipophilic agent(e.g., a Wnt protein, such as Wnt3A) inserted in the non-aqueous phaseof a lipid structure (e.g., L-WNT3A) is applied to the dentin (or insome cases applied to exposed pulp). In other words, in some cases,dentin and/or exposed pulp is contacted with a Wnt stimulatingcomposition and/or a lipophilic agent (e.g., a Wnt protein, such asWnt3A) inserted in the non-aqueous phase of a lipid structure. A subjectcomposition can be re-applied over the course of 1 to 30 minutes (e.g.,1 to 15 minutes, 2 to 15 minutes, 2 to 12 minutes, 4 to 15 minutes, 4 to12 minutes, 5 to 15 minutes, 5 to 12 minutes, 5 to 10 minutes, 7 to 15minutes, 7 to 12 minutes, or 8 to 12 minutes). In some cases, aftercontacting the denting and/or exposed pulp with a subject composition,the tooth is closed using standard tooth replacement materials(composite resin, amalgam, glass ionomer cement, etc). In some cases, asubject method includes a step of closing the tooth. In some cases,dentin and/or exposed pulp is contacted with a subject composition twomore times (e.g, 3 or more times, 4 or more times, 5 or more times) overa period of time in a range of from 1 to 30 minutes (e.g., 1 to 15minutes, 2 to 15 minutes, 2 to 12 minutes, 4 to 15 minutes, 4 to 12minutes, 5 to 15 minutes, 5 to 12 minutes, 5 to 10 minutes, 7 to 15minutes, 7 to 12 minutes, or 8 to 12 minutes).

The dosage of the therapeutic formulation will vary widely, dependingupon the nature of the condition, the frequency of administration, themanner of administration, the clearance of the agent from the host, andthe like. The initial dose can be larger, followed by smallermaintenance doses. The dose can be administered as infrequently asweekly or biweekly, or more often fractionated into smaller doses andadministered daily, semi-weekly, or otherwise as needed to maintain aneffective dosage level.

In some embodiments, administration of a subject composition (e.g., awnt stimulator composition, a composition that includes a lipohilicagent, etc.) is performed by local administration. Local administration,as used herein, may refer to topical administration, but can also referto injection or other introduction into the body at a site of treatment(e.g., at or near the sight of a dental injury, tooth sensitivity,etc.).

In some embodiments, a subject composition is administered on a shortterm basis, for example a single administration, or a series ofadministration performed over, e.g. 1, 2, 3 or more days, up to 1 or 2weeks. The size of the dose administered can be determined by aphysician and will depend on a number of factors, such as the nature andgravity of the disease, the age and state of health of the patient andthe patients tolerance to the procedure and/or to the composition.

The terms “co-administration”, “co-administer”, and “in combinationwith” include the administration of two or more therapeutic agents(e.g., a subject Wnt stimulating composition and/or a subject lipophilicagent inserted in the non-aqueous phase of a lipid structure; and/oranother dentin-stimulating agent, a pulp survival agent, ananti-infection agent, and the like) either simultaneously, concurrentlyor sequentially within no specific time limits. In one embodiment, theagents are contacted with the target tissue (e.g., pulp and/or dentin)or in the subject's body at the same time or exert their biological ortherapeutic effect at the same time. In some embodiments, thetherapeutic agents are in the same composition or unit dosage form. Insome embodiments, the therapeutic agents are in separate compositions orunit dosage forms. In certain embodiments, a first agent can beadministered prior to (e.g., minutes, 15 minutes, 30 minutes, 45minutes, 1 hour, 2 hours, 4 hours, 6 hours, 12 hours, 24 hours, 48hours, 72 hours, 96 hours, 1 week, 2 weeks, 3 weeks, 4 weeks, 5 weeks, 6weeks, 8 weeks, or 12 weeks before), concomitantly with, or subsequentto (e.g., 5 minutes, 15 minutes, 30 minutes, 45 minutes, 1 hour, 2hours, 4 hours, 6 hours, 12 hours, 24 hours, 48 hours, 72 hours, 96hours, 1 week, 2 weeks, 3 weeks, 4 weeks, 5 weeks, 6 weeks, 8 weeks, or12 weeks after) the administration of a second therapeutic agent.

In some cases, a subject Wnt stimulating composition and/or a subjectlipophilic agent inserted in the non-aqueous phase of a lipid structure(e.g., formulated as a pharmaceutical composition) is co-administeredwith one or more additional agents (e.g., a dentin-stimulating agent, apulp survival agent, an anti-infection agent, and/or a growth factor,etc.). Such administration may involve concurrent (i.e. at the sametime), prior, or subsequent administration of the additional agent withrespect to the administration of an agent or agents of the disclosure.In some embodiments, treatment is accomplished by administering acombination (co-administration) of a subject Wnt stimulating compositionand/or a subject lipophilic agent inserted in the non-aqueous phase of alipid structure with another agent (e.g., a dentin-stimulating agent, apulp survival agent, an anti-infection agent, and/or a growth factor,etc.).

Treatment may be combined with other active agents, such as antibiotics,cytokines, etc. Classes of antibiotics include penicillins, e.g.penicillin G, penicillin V, methicillin, oxacillin, carbenicillin,nafcillin, ampicillin, etc.; penicillins in combination with β-lactamaseinhibitors, cephalosporins, e.g. cefaclor, cefazolin, cefuroxime,moxalactam, etc.; carbapenems; monobactams; aminoglycosides;tetracyclines; macrolides; lincomycins; polymyxins; sulfonamides;quinolones; cloramphenical; metronidazole; spectinomycin; trimethoprim;vancomycin; etc. Cytokines may also be included, e.g. interferon γ,tumor necrosis factor α, interleukin 12, etc.

A “therapeutically effective dose” or “therapeutic dose” is an amountsufficient to effect desired clinical results (i.e., achieve therapeuticefficacy). A therapeutically effective dose can be administered in oneor more administrations. For purposes of this disclosure, atherapeutically effective dose of a subject Wnt stimulating compositionand/or a subject lipophilic agent inserted in the non-aqueous phase of alipid structure is an amount that is sufficient to palliate, ameliorate,stabilize, reverse, prevent, slow or delay the progression of thedisease and/or injury state (e.g., pulp exposure, tooth sensitivity,etc.). Thus, a therapeutically effective dose of a subject Wntstimulating composition and/or a subject lipophilic agent inserted inthe non-aqueous phase of a lipid structure can increase the amount ofdentin produced by the tooth pulp, can decrease cell death in the toothpulp (e.g., which can be detected using techniques such as TUNELstaining, Casp8 expression, CASPASE 3 expression, and the like); canincrease cell proliferation in the tooth pulp (e.g., which can bedetected using techniques such as Ki67 immunostaining, BrdUincorporation, and the like); can increase the number of differentiatedodontoblasts in the tooth pulp (e.g., which can be detected usingexpression markers such as Nestin and/or the extracellular matrixprotein DSP); can increase levels of highly organized tubular dentinmatrix in the tooth pulp; and can increase levels of a collagenousmatrix with a linear organization (suggestive of tubular orthodentin) inthe tooth pulp (e.g., as can be detected by picrosirius red staining andpolarized light).

As such, in some cases, the subject methods include a step of evaluatingtooth pulp for increased dentin produced by the tooth pulp, decreasedcell death in the tooth pulp (e.g., which can be detected usingtechniques such as TUNEL staining, Casp8 expression, CASPASE 3expression, and the like); increased cell proliferation in the toothpulp (e.g., which can be detected using techniques such as Ki67immunostaining, BrdU incorporation, and the like); increased number ofdifferentiated odontoblasts in the tooth pulp (e.g., which can bedetected using expression markers such as Nestin and/or theextracellular matrix protein DSP); increased levels of highly organizedtubular dentin matrix in the tooth pulp; and/or increased levels of acollagenous matrix with a linear organization (suggestive of tubularorthodentin) in the tooth pulp (e.g., as can be detected by picrosiriusred staining and polarized light). The “increase” and/or “decrease” canbe relative to any convenient control (e.g., a pre-determined value, anuntreated control tooth, a sample from the same patient evaluated priorto treatment; a control treated with a placebo (e.g., a salinesolution); and the like).

In some cases, the subject methods include a step of evaluating whethera treatment stimulated (e.g., increased) Wnt signaling (i.e., activityof the Wnt signaling pathway). Any convenient method can be used todetect such activity (e.g., expression of a target gene of the Wntsignaling pathway, increase of a Wnt reporter, etc.). The “increase”and/or “decrease” can be relative to any convenient control (e.g., apre-determined value, an untreated control tooth, a sample from the samepatient evaluated prior to treatment; a control treated with a placebo(e.g., a saline solution); and the like).

It will be understood by one of skill in the art that guidelines forparameters such as dosage and frequency can be adjusted for variousfactors such as molecular weight of the active agent, type ofadministration, e.g. intranasal, inhalation, topical, injection,systemic (e.g. i.m., i.p., i.v.), and the like. A subjectcomposition/agent can be administered by any suitable means, includingtopical, oral, parenteral, intrapulmonary, and intranasal. Parenteralinfusions include intramuscular, intravenous (bollus or slow drip),intraarterial, intraperitoneal, intrathecal or subcutaneousadministration. Administration may include injection, parenteral routessuch as intravenous, intravascular, intraarterial, subcutaneous,intramuscular, intratumor, intraperitoneal, intraventricular,intraepidural, or others as well as oral, nasal, ophthalmic, rectal, ortopical. Sustained release administration is also specifically includedin the disclosure (e.g., such as depot injections or erodible implants).Localized delivery is contemplated, e.g., topical administration todentin and/or contact with exposed pulp tissue.

As noted above, a subject composition/agent can be formulated with apharmaceutically acceptable carrier (one or more organic or inorganicingredients, natural or synthetic, with which a subject agent iscombined to facilitate its application). A suitable carrier includessterile saline although other aqueous and non-aqueous isotonic sterilesolutions and sterile suspensions known to be pharmaceuticallyacceptable are known to those of ordinary skill in the art. An“effective amount” refers to that amount which is capable ofameliorating or delaying progression of the diseased, degenerative ordamaged condition. An effective amount can be determined on anindividual basis and will be based, in part, on consideration of thesymptoms to be treated and results sought.

Kits

Also provided are kits for use in the methods (e.g., pharmaceutical packor kit including one or more containers having one or more of theingredients of the pharmaceutical compositions of the invention. Thesubject kits can include a Wnt stimulating composition that includes aWnt stimulator agent (e.g., at a dose sufficient to enhance theproduction of dentin by dental pulp) and/or a lipophilic agent insertedin the non-aqueous phase of a lipid structure. In some cases, a Wntstimulator agent is a lipophilic agent inserted in the non-aqueous phaseof a lipid structure. In some embodiments, a kit comprises two or Wntstimulator agents and/or two or more lipophilic agents inserted in thenon-aqueous phase of a lipid structure (e.g., two or more lipophilicagents each inserted in the non-aqueous phase of separate lipidstructures; and/or two or more lipophilic agents inserted in thenon-aqueous phase of the same lipid structure). In some embodiments, aWnt stimulator agent and/or a lipophilic agent inserted in thenon-aqueous phase of a lipid structure is provided in a dosage form(e.g., a therapeutically effective dosage form). In the context of akit, a Wnt stimulator agent and/or a lipophilic agent inserted in thenon-aqueous phase of a lipid structure can be provided in liquid or soldform in any convenient packaging (e.g., stick pack, dose pack, etc.).The agents of a kit can be present in the same or separate containers.For example, a kit may have a Wnt stimulator agent in one container andin another container, a lipophilic agent inserted in the non-aqueousphase of a lipid structure. As another example, a kit may have a Wntstimulator agent in one container and in another container, another Wntstimulatory agent. As yet another example, a kit may have in onecontainer a lipophilic agent inserted in the non-aqueous phase of alipid structure, and in another container, a different a lipophilicagent inserted in the non-aqueous phase of a lipid structure. In somecases, the agents of a subject kit are present in the same container.The above kits may include a reagent and/or component for a dentalprocedure associated with the subject methods (e.g., a reagent and/orcomponent for capping a tooth, for exposing dentin, for exposing pulp,and the like).

In addition to the above components, the subject kits may furtherinclude (in certain embodiments) instructions for practicing the subjectmethods. These instructions may be present in the subject kits in avariety of forms, one or more of which may be present in the kit. Oneform in which these instructions may be present is as printedinformation on a suitable medium or substrate, e.g., a piece or piecesof paper on which the information is printed, in the packaging of thekit, in a package insert, and the like. Yet another form of theseinstructions is a computer readable medium, e.g., diskette, compact disk(CD), flash drive, and the like, on which the information has beenrecorded. Yet another form of these instructions that may be present isa website address which may be used via the internet to access theinformation at a removed site.

The invention now being fully described, it will be apparent to one ofordinary skill in the art that various changes and modifications can bemade without departing from the spirit or scope of the invention.

EXPERIMENTAL

The following examples are put forth so as to provide those of ordinaryskill in the art with a complete disclosure and description of how tomake and use the present invention, and are not intended to limit thescope of what the inventors regard as their invention nor are theyintended to represent that the experiments below are all or the onlyexperiments performed. Efforts have been made to ensure accuracy withrespect to numbers used (e.g. amounts, temperature, etc.) but someexperimental errors and deviations should be accounted for. Unlessindicated otherwise, parts are parts by weight, molecular weight isweight average molecular weight, temperature is in degrees Centigrade,and pressure is at or near atmospheric.

All publications and patent applications cited in this specification areherein incorporated by reference as if each individual publication orpatent application were specifically and individually indicated to beincorporated by reference.

The present invention has been described in terms of particularembodiments found or proposed by the present inventor to comprisepreferred modes for the practice of the invention. It will beappreciated by those of skill in the art that, in light of the presentdisclosure, numerous modifications and changes can be made in theparticular embodiments exemplified without departing from the intendedscope of the invention. For example, due to codon redundancy, changescan be made in the underlying DNA sequence without affecting the proteinsequence. Moreover, due to biological functional equivalencyconsiderations, changes can be made in protein structure withoutaffecting the biological action in kind or amount. All suchmodifications are intended to be included within the scope of theappended claims.

EXAMPLES Example 1

The experiments below show a liposome-reconstituted form of WNT3Aproduces a stable form of the protein that when used as a biomimeticcompound activates cells in the injured tooth pulp, stimulating dentinregeneration. A Wnt-amplified environment was found to be associatedwith superior pulp healing after pulp injury, for example, the number ofcells undergoing apoptosis was significantly reduced, resulting insignificantly better survival of injured odontoblasts and an increase intertiary dentin. Pulp cells responded to elevated Wnt stimulus bydifferentiating into dentin-secreting odontoblasts. Thus, transientlyamplifying a Wnt response resulted in improved pulp vitality.

Materials and Methods

Animals

The Stanford Committee on Animal Research and the Animal Care Committeeof the University Paris Descartes (agreement CEEA34.CC.016.11, Comitéd'éthique pour l'expérimentation animale no. 34, Paris, France) approvedall experimental procedures. Rats were purchased from Janvier Labs.Axin2^(LacZ/LacZ) (#11809809) and Axin2^(CreERT2/+); R26R^(mTmG/+)(#018867 and #007576, respectively) mice were purchased from JacksonLabs. For Axin2^(CreERT2/+); R26R^(mTmG/+) mice, tamoxifen was deliveredIP (4.0 mg/25 mg body weight) for 5 consecutive days.

Animal Surgeries

Adult male mice (3-5 months old) were anesthetized with anintraperitoneal injection of ketamine (80 mg/kg) and xylazine (16mg/kg). In total, 72 mice (36 Axin2^(LacZ/+) and 36 Axin2^(LacZ/LacZ)mice) were used. A cavity was created with a Ø 0.3 mm diameter round bur(E0123, Dentsply Maillefer, Ballaigues, Switzerland) then a #6 k-filewas used to expose the dental pulp. Glass ionomer cement (3M) was usedto cap the injury. Mice were sacrificed at times indicated in theexperiments.

In rats, a cavity was created with a Ø 0.2-mm-diameter round bur then aroot-canal-shaping rotary nickel-titanium file system (Protaper,Dentsply) was used to expose the dental pulp. After pulp exposure, beadstreated either with L-WNT3A (N=18), or L-PBS (N=18) were implanted intothe pulp chamber using a blunt steel probe (see below for details on thepreparation of the beads). Biodentine cement (Septodont, Saint-Maur desFosse's, France) was used to cap the injury. Rats were sacrificed attimes indicated in the experiments.

Preparation and Delivery of L-WNT3A

Purified recombinant human WNT3A protein was incubated with liposomevesicles as described (13). L-WNT3A (10 ng, see (14)) or L-PBS (N=18 foreach condition) was delivered on Affi-Gel agarose beds (Bio-RadLaboratories) that had been soaked overnight at 37° C. in the relevantsolutions (15).

Sample Preparation, Processing, Histology, Histomorphometrics, andCellular Assays

Maxillae were harvested, the skin and outer layers of muscle wereremoved, and the tissues were fixed. Tissues were sectioned at athickness of 8 μm and processed using established procedures (16).Histologic staining was performed as described (16). A minimum of sixsections were used to quantify the amount of new dentin.Histomorphometric measurements were performed as described (17).

X-gal staining was performed as described (18). TUNEL staining wasperformed as described by the manufacturer (In Situ Cell Death DetectionKit, Roche). Immunostaining was performed using standard procedures(10). For cell proliferation analysis, BrdU labeling reagent(Invitrogen, Carlsbad, Calif.) was either injected IP, or added toculture media according to the manufacturer's instructions; animals weresacrificed 12 hours later and both bone marrow-derived stem cells anddental pulp cells were fixed 12 hours later.

Primary antibodies and their dilutions were as follows:anti-biotinylated BrdU (1:200), anti-Nestin (1:300), anti-Ki67 (1:200),anti-PCNA (1:1000), anti-DSP (1:1000).

Dental Pulp Stem Cells and Bone Marrow Treatments

Human dental pulp stem cells were isolated as described (19), inaccordance with the Ethics committee of the Pirkanmaa Hospital District,Tampere, Finland (R06009). Cells were cultured in Dulbecco modifiedEagle Medium (DMEM) containing Nutrient Mixture F-12 with 10% fetalbovine serum. Cells were treated with L-PBS or L-WNT3A (effectiveconcentration=0.06 μg/mL) at 37° C. for 6, 12, and 24 hours. RNA wasisolated afterwards and analyzed by qRT-PCR (see below) and BrdUincorporation (below).

Bone marrow was harvested from the femora and tibiae of adult mice,aliquoted to produce similar sized samples. DNA content was measured toensure that variation between samples was <10% (20). Each aliquot wasincubated with 20 μL of DMEM with 10% fetal bovine serum containingL-PBS or L-WNT3A (effective concentration=0.15 μg/mL) at 37° C. for 4hours. RNA was isolated afterwards and analyzed by qRT-PCR, or thetissues were fixed in 4% PFA at 4° C. then processed into OCT forcryosectioning. TUNEL activity and Ki67 expression were analyzed using10 μm sections (see above).

Quantitative RT-PCR

Total RNA was extracted using TRIzol (Invitrogen). cDNA was synthesizedby using SuperScript III First-Strand Synthesis Kit (Invitrogen)according to the instructions of the manufacturer. RT-PCR andquantitative PCR (ABI Prism 7900 HT Sequence Detection System) wereperformed as described (10). All reactions were performed in triplicate.

The following primer sets were used:

Axin2, (SEQ ID NO: 1) 5′-ACCCTGGGCCACTTTAAAG-3′ (sense) and(SEQ ID NO: 2) 5′-CCTTCATACATCGGGAGCAC-3′ (antisense); Axin2 exon 1,(SEQ ID NO: 3) 5′-TCAGTAACAGCCCAAGAACC-3′ (sense) and (SEQ ID NO: 4)5′-GAGCCTCCTCTCTTTACAGC-3′ (antisense); CASP3, (SEQ ID NO: 5)5′-GCACTGGAATGTCATCTCGCT-3′ (sense) and (SEQ ID NO: 6)5′-GGCCCATGAATGTCTCTCTGAG-3′ (antisense); Lef1, (SEQ ID NO: 7)5′-ACACCCTGATGAAGGAAAGC-3′ (sense) and (SEQ ID NO: 8)5′-GACCCATTTGACATGTACGG-3′ (antisense); PCNA, (SEQ ID NO: 9)5′-CTTGGAATCCCAGAACAGGA-3′ (sense) and (SEQ ID NO: 10)5′-CAGCATCTCCAATGTGGCTA-3 (antisense); Nestin: (SEQ ID NO: 11)5′-CTCGGGAGAGTCGCTTAGAG-3′ (sense) and (SEQ ID NO: 12)5′-CACAGCCAGCTGGAACTTT-3′ (antisense);Dentin sialophosphoprotein (DSPP): (SEQ ID NO: 13)5′-GGAATGGAGAGAGGACTGCT-3′ (sense) and (SEQ ID NO: 14)5′-AGGTGTTGTCTCCGTCAGTG-3′ (antisense); Osteocalcin: (SEQ ID NO: 15)5′-TGTGACGAGCTATCAAACCAG-3′ (sense) and (SEQ ID NO: 16)5′-GAGGATCAAGTTCTGGAGAGC-3′ (antisense); and Collagen type I:(SEQ ID NO: 17) 5′-AAGGACAAGAGGCACGTCTG-3′ (sense) and (SEQ ID NO: 18)5′-CGCTGTTCTTGCAGTGGTAG-3′ (antisense).

Dentin Volume and Mineral Density Micro-CT Analysis

Micro-computed topographies of the maxillae were performed using aSkyScan 1176 scanner (SkyScan, Bruker, Belgium) at a 5 μm resolution.Scanning was done at 45 kV, 556 mA. Reconstruction of sections wasachieved using a modified Feldkamp cone-beam algorithm with beamhardening correction set to 50%. CTAnalyzer software (version 1.02;SkyScan) was employed for morphometric quantification.

Reparative Dentin Histomorphometry

Sections from rat molars were examined morphometrically at a constantmagnification (250×) with a semi-automatic image analyzer coupling themicroscope to a video camera and a computer (21). Six sections persample (N=6 molars per group) were taken at the center of the pulpexposure site. At day 14, the porosity of the dentin bridge wasdetermined on Masson's trichrome-stained sections by measuring thepercentage of space containing cells within the reparative dentin.

Statistical Analyses

Results are presented as mean±standard error values of independentreplicates.

Student's t test was used to quantify differences described in thisarticle. P0.05 was considered to be significant.

Results

When Odontoblasts are Wnt Responsive

Odontoblasts are distinguished from pulp cells by the expression of theintermediate filament protein Nestin (FIG. 1C (22)), and dentinsialoprotein, DSP (FIG. 1A-D) (23)). Using X-gal staining of tissuesfrom Axin2^(LacZ/+) mice, in which the promoter of the Wnt target geneAxin2 drives LacZ expression (24,25), the odontoblasts lining the innersurface of the pulp cavity were Wnt responsive (FIG. 1E). A second,inducible Axin2 reporter strain (Axin2^(CreERT2/+); R26R^(mTmG/+))verified that odontoblasts respond to an endogenous Wnt signal: GFPimmunofluorescence was readily apparent in odontoblast cell bodies andthe processes that extended into the dentin (FIG. 1F). Analyses ofembryonic and early post-natal dental tissues (FIG. 6) showed thatodontoblasts were also Wnt responsive, indicating that these cellsmaintain a Wnt responsive status throughout their lifetime.

Deletion of Axin2 does not Affect the Dentin/Pulp Complex

Using Axin2^(LacZ/LacZ) mice, in which the negative Wnt regulator Axin2is deleted (24,25), and Wnt responsiveness is elevated (10,26), thegross morphology of the teeth from Axin2^(LacZ/+) and Axin2^(LacZ/LacZ)mice was evaluated and no significant differences were found in the sizeof the pulp cavities, or the thickness and density of the alveolar bone,and the size of the pulp chambers was unaffected by Axin2 deletion (FIG.2A-F). The dentin volume (FIG. 2G), and the mineral densities of enameland dentin were also equivalent in Axin2^(LacZ/+) and Axin2^(LacZ/LacZ)mice (FIG. 2H). Histologic examination showed that the Axin2^(LacZ/LacZ)pulp was indistinguishable from heterozygous and wild-type littermates(FIG. 2I,J; N≥20 for each genotype). The distribution of X-gal^(+ve)cells in Axin2^(LacZ/LacZ) and Axin2^(LacZ/+) mice was unchanged; theonly difference of note was the intensity of X-gal staining inAxin2^(LacZ/LacZ) mice, which is expected since the homozygous micecarry two copies of the LacZgene (FIG. 2K, L).

Axin2 is a ligand-dependent inhibitor of Wnt signaling; consequently, itis anticipated that in the absence of a Wnt stimulus Axin2^(Lac/LacZ)mice should show baseline Wnt signaling, equivalent to that seen inAxin2^(LacZ/+) and wild type mice (10,24). Quantitative RT-PCR verifiedthat baseline Wnt signaling, as measured by Lef1 and Axin2 (exon 1)expression was equivalent in Axin2^(LacZ/+) and Axin2^(LacZ/LacZ) mice(FIG. 2M). Markers of cell proliferation (FIG. 2M), and the odontogenicproteins Nestin (FIG. 2N,O), DSPP, Osteocalcin, and Collagen type I(FIG. 2P) showed no significant differences in expression levels betweenAxin2^(LacZ/+) and Axin2^(LacZ/LacZ) mice.

Axin2^(LacZ/LacZ) Mice Exhibit a Superior Reparative Response FollowingAcute Pulp Exposure

The response of Axin2^(LacZ/LacZ) mice and their Axin2^(LacZ/+) controllittermates to an acute pulp exposure was tested. By post-op day 14, thepulp cavities in Axin2^(LacZ/+) mice were largely necrotic (N=6; FIG.3A). A distinctly different response was observed in Axin2^(LacZ/LacZ)mice, where instead of necrotic pulp tissue the cavity was occupied byreparative dentin (N=6; FIG. 3B; quantified in C). The organization ofthis matrix was examined using picrosirius red staining and withvisualization under polarized light. In Axin2^(LacZ/+) controls, noorganized collagenous network was evident in the injury site (FIG. 3D);in contrast, in Axin2^(LacZ/LacZ) mice a dense and packed collagen fibernetwork forming the bridge was obvious (FIG. 3E). In Axin2^(LacZ/LacZ)mice but not in controls, dentin-secreting cells were immunopositive forDSP (FIG. 3G) and Nestin (FIG. 3I).

On post-op day 4, granulation tissue filled the pulp chambers inAxin2^(LacZ/+) controls (N=6; FIG. 3J). Axin2^(LacZ/LacZ) mice showedminimal granulation tissue (N=6; FIG. 3K). Quantitative RT-PCR revealedthat the endogenous Wnt response, as measured by Axin2 exon1 expression,was significantly elevated in Axin2^(LacZ/LacZ) mice compared tocontrols (FIG. 3L).

Exposure of the pulp causes extensive cellular necrosis (28); there isalso a period of latent apoptosis when pulp cells damaged by the injurycan either die or recover (29). In Axin2^(LacZ/+) controls, abundantTUNEL staining identified these dying cells (FIG. 3M). InAxin2^(LacZ/LacZ) mice, very few TUNEL^(+ve) cells were evident, even onpost-op day 4 (FIG. 3N). Apoptosis is largely controlled by caspaseactivity (30) and as anticipated by the TUNEL staining, Casp8 expressionin Axin2^(LacZ/LacZ) mice was significantly lower than its expression inAxin2^(LacZ/+) controls (FIG. 3O). Cell proliferation, as indicated byKi67 immunostaining, was greater in Axin2^(LacZ/LacZ) mice compared toAxin2^(LacZ/+) controls (FIG. 3P,Q). Thus, in response to an acute pulpinjury that caused a significant elevation in endogenous Wnt signaling,Axin2^(LacZ/LacZ) mice fared better than their heterozygous littermates.The elevated Wnt environment was correlated with reduced cell death,enhanced cell proliferation, and an overall improvement in the repairresponse of the pulp.

Wnt Signaling Regulates Apoptosis and Proliferation in Dental Pulp StemCells

It was tested whether a Wnt stimulus alone was sufficient to reduce celldeath and enhance cell proliferation in pulp cells. Dental pulp stemcells were isolated from human teeth (19) and analyzed first for theirresponsiveness to WNT3A protein (13). Within 6 hours of treatment,dental pulp stem cells exhibited a 4.8-fold increase in Axin2 expressionthat persisted for at least 24 h (FIG. 4A). The mitotic activity ofdental pulp stem cells was significantly increased by L-WNT3A treatment(FIG. 4B). Human CASPASE 3 expression was significantly reduced byL-WNT3A treatment (FIG. 4C).

In their undifferentiated state, pulp and bone marrow have beenconsidered equivalent tissues (31,32). Whether freshly harvested bonemarrow responded to L-WNT3A in a manner similar to the human dental pulpstem cells was then tested. Whole bone marrow from mice was harvestedand treated with L-WNT3A or L-PBS and within 24 h a significant increasein Wnt responsiveness was detected (FIG. 4D). The elevation in Wntresponsiveness occurred simultaneous with an increase in cellproliferation (FIG. 4E) and a reduction in cell death (FIG. 4F). Thus,exposure to a WNT stimulus is sufficient to activate Wnt signaling,enhance mitotic activity, and reduce apoptosis in two stem cellpopulations.

L-WNT3A Treatment Preserves Pulp Vitality after an Acute Pulp Exposure

Given the ability of L-WNT3A to reduce apoptosis and promote cellproliferation in vitro, it was then tested whether L-WNT3A could elicitsimilar effects in pulp tissue after an acute injury. Acute pulpexposures were generated in wild-type rats and treated with L-WNT3A or aliposomal formulation of PBS (L-PBS) then sealed to prevent bacterialcontamination. Histological analyses verified that the size and extentof the injury was equivalent between the treatment groups (N=6 for bothtreatment groups; FIG. 5A,B).

By post-op day 4, L-PBS controls exhibited extensive pulp necrosis andapoptosis (N=6, FIG. 5C,C′); in L-WNT3A treated cases, TUNEL stainingwas minimal (N=6; FIG. 5D). The TUNEL staining that was observed in theL-WNT3A treated samples was generally restricted to the roof of the pulpcavity, near to the site of exposure (N=6; FIG. 5D′).

In an elevated Wnt environment such as is observed in Axin2^(LacZ/LacZ)mice, cell proliferation is significantly elevated after an injury (FIG.3); this suggests a more vigorous repair response. The same effectfollowing L-WNT3A treatment of the injured pulp was observed: relativeto L-PBS treated pulp exposures, proliferating cell nuclear antigen(PCNA) immunostaining was much more extensive in the L-WNT3A treatedsamples (compare FIG. 5E,F).

Reduced apoptosis and increased proliferation in the L-WNT3A treatedpulps culminated in a superior repair response. In L-PBS cases, the pulpwas largely occupied on post-op day 14 by a amorphous, bone-like tissue,called atubular osteodentin ((33); FIG. 5G) whereas in L-WNT3A treatedcases the pulp cavities were filled with a highly organized, tubulardentin matrix (FIG. 5H). Similar to previous quantitative analyses (FIG.3C) the dentin appeared to be denser in the L-WNT3A treated samplescompared to the L-PBS controls (FIG. 5I).

The reparative dentin matrix, as visualized by picrosirius red stainingand polarized light, was distinctly different between the two groups: inL-PBS samples the collagenous matrix exhibited a basket-weaved pattern,characteristic of bone (FIG. 5J); in L-WNT3A samples, the collagenousmatrix had a linear organization, suggestive of tubular orthodentin(FIG. 5K).

Differentiated odontoblasts express Nestin (34); few Nestin^(+ve) cellswere detected in L-PBS treated samples compared to the L-WNT3A samples(FIG. 5L,M). Differentiated odontoblasts also express the extracellularmatrix protein DSP (23) and DSP^(+ve) cells were largely absent from theL-PBS treated pulp compared to the L-WNT3A treated pulp (FIG. 5N,O).

DISCUSSION

When confronted with noxious stimuli the human pulp is capable ofmounting a robust repair response—at least in young patients (35,36). Inolder individuals, the pulp responds to the same noxious stimuli byundergoing necrosis (35).

Previous reports indicated that after postnatal day 15, molarodontoblasts and odontoblasts at the incisor tip lose their Wntresponsiveness (44). The question of whether polarized, secretoryodontoblasts maintain their dependence upon a Wnt signal into adulthoodwas addressed herein. Two separate approaches were used: first,cryo-sectioned tissues from adult Axin2^(LacZ/+) Wnt reporter mice wereanalyzed and both polarized odontoblasts and pulp cells were found to beX-gal^(+ve) (FIG. 1E, and FIG. 2K). Second, tissues from adultAxin2^(CreERT2/+); R26R^(mTmG/+) Wnt reporter mice also illustrated thatpolarized odontoblasts were GFP^(+ve) (FIG. 1F). Thus, adultodontoblasts and pulp cells maintain a Wnt responsive status inadulthood.

In the absence of a Wnt stimulus Axin2^(LacZ/LacZ) cells behave the sameas wild-type cells (10). Because Axin2 represses Wnt signaling in aligand-dependent manner (24,25), the removal of Axin2 results in anamplified Wnt response (10,24). The response to pulpal injury can beenhanced by elevating Wnt signaling by either removing a negative Wntregulator (FIG. 3), or by providing exogenous WNT3A protein (FIG. 5),which is sufficient to significantly improve the pulp cavity's repairresponse.

The mechanism of WNT action in the pulp may be due in part to theresponse of stem/progenitor cells within this tissue. Human dental pulpstem cells responded to human WNT3A protein by strongly up activatingthe Wnt pathway, by becoming mitotically active, and by down-regulatingcaspase activity (FIGS. 4,5), an enzyme that mediates the executionphase of apoptosis (50). Collectively, these biological responses arevaluable in therapeutic strategies that seek to improve a healingresponse.

In addition to these biological responses, a difference was noted in thetype of reparative mineralized tissue that formed after L-PBS andL-WNT3A treatment (FIG. 5). In L-PBS treated samples, a bone-likemineralized matrix, osteodentin, forms. Compared to dentin, osteodentinis porous and consequently its appearance in the injured pulp representsa sub-optimal healing response. In L-WNT3A cases the reparative matrixresembled native dentin (FIG. 5H,K). This dentin matrix was produced bynative DSP^(+ve) secretory odontoblasts (FIG. 5M) and it formed a dentinbridge that effectively separated the viable pulp cavity from theexternal environment. No such dentin bridge was evident in controls.

The present disclosure provides an approach (e.g., to root canaltherapy) that exploits the reliance of pulp cells on endogenous Wntsignaling (FIGS. 3,4). A liposome-reconstituted form of WNT3A proteineffectively protected pulp cells from death and stimulated proliferationof undifferentiated cells in the pulp, which together significantlyimproved pulp healing. The strategy of activating endogenous stem cellsvia L-WNT3A to improve healing represents a viable means to achievingpulp regeneration in humans.

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Example 2

The example provided herein demonstrates penetration of L-Wnt3A throughdentinal tubules and activation of pulp cells.

Rodent dentinal tubules and human dentinal tubules have similardiameters.

Therefore, a rodent model was used to demonstrate penetration of theL-WNT3A through the dentinal tubules to the pulp (FIG. 8). Topicalapplication of the liposomal mixture penetrates the dentinal tubules. Ina Wnt reporter Axin2CreERT2/+; R26RmTmG/+ mouse strain, the delivery ofthe hydrophobic tamoxifen molecule, in association with the sameliposome used to package WNT3A, results in Cre-mediated recombinationevent in pulp cells located immediately beneath the cavity preparation.L-WNT3A exposure acts as a survival signal for odontoblasts. Typicallyafter exposure to a toxic (e.g., heat, chemical) agent, mostodontoblasts die; this eventually necessitates the removal of thenecrotic pulp and its replacement with an inert filling material,through a process known as a root canal. Cell death is significantlydecreased provided odontoblasts are exposed to the pro-survival signalL-WNT3A.

Liposomes were fabricated from dimyristoyl-phosphatidylcholine lipids.Liposomes were made with tamoxifen and used in combination with atamoxifen inducible mouse strain Axin2CreERT2/+; R26RmTmG/+ and theirpenetration were monitored over the course of 14 d. The liposomes wereeffective at penetrating to the cells below the deep cavity preparationand inducing genetic recombination of the cell. The ability of L-WNT3Ato stimulate proliferation in dental pulp stem and progenitor cells, andin odontoblasts was demonstrated above. The ability of L-WNT3A tocurtail programmed cell death through a caspase 8-mediated mechanism wasdemonstrated above. The ability of L-WNT3A to enhance expression ofmolecules associated with dentin secretion including dentin sialoprotein(DSP) and Nestin has also been demonstrated herein.

What is claimed:
 1. A method for enhancing dentin production, the methodcomprising: (a) providing a tissue from a subject; (b) contacting thetissue with liposomal Wnt, wherein the liposomal Wnt comprises a Wntprotein, or a fragment thereof, inserted a non-aqueous phase of a lipidstructure, thereby forming a Wnt-modified tissue; and (c) administeringthe Wnt-modified tissue to a tooth of the subject.
 2. The methodaccording to claim 1, wherein the Wnt protein is human Wnt3A.
 3. Themethod according to claim 1, wherein the human Wnt3A protein has anamino acid sequence of SEQ ID NO:
 19. 4. The method according to claim1, wherein the lipid structure comprises a size range of from 0.03 to0.2 micron.
 5. The method according to claim 1, wherein the tissue isderived from dental pulp.
 6. The method according to claim 1, whereinthe tissue is derived from bone marrow.
 7. The method according to claim1, wherein the tissue provided from the subject is from the tooth towhich the Wnt-modified tissue is administered.
 8. The method accordingto claim 1, wherein the tissue provided from the subject is from adifferent tooth of the subject than that to which the Wnt-modifiedtissue is administered.
 9. The method according to claim 1, wherein theWnt-modified tissue is present in an amount sufficient to activate stemcells, progenitor cells, or a combination thereof in the tissue.
 10. Themethod according to claim 1, wherein the Wnt-modified tissue is presentin an amount sufficient to reduce apoptosis of a dental pulp tissue inthe tooth of the subject.
 11. The method according to claim 1, whereinthe Wnt-modified tissue is administered to dental pulp tissue within thetooth.
 12. The method according to claim 1, wherein administering theWnt-modified tissue increases the number of differentiated odontoblastsin the tooth.
 13. The method according to claim 1, wherein theWnt-modified tissue promotes secretion of dentin matrix fromodontoblasts in the tooth.
 14. The method according to claim 1, whereinthe Wnt-modified tissue further comprises a pharmaceutically acceptablecarrier.
 15. The method according to claim 1, wherein the Wnt-modifiedtissue is administered as a single administration.
 16. The methodaccording to claim 1, wherein the Wnt-modified tissue is administered aspart of a series of administrations.
 17. The method according to claim1, wherein the subject is a human.
 18. The method according to claim 1,wherein the method is performed as part of a dental procedure.
 19. Themethod according to claim 18, wherein the dental procedure is a deepdental restoration.
 20. The method according to claim 19, wherein thedeep dental restoration comprises placement of an amalgam, composite, orcrown within the tooth.